Soybean seed and oil compositions and methods of making same

ABSTRACT

Methods for obtaining soybean plants that produce seed with low linolenic acid levels and moderately increased oleic levels are disclosed. Also disclosed are methods for producing seed with low linolenic acid levels, moderately increased oleic levels and low saturated fatty acid levels. These methods entail the combination of transgenes that provide moderate oleic acid levels with soybean germplasm that contains mutations in soybean genes that confer low linolenic acid phenotypes. These methods also entail the combination of transgenes that provide both moderate oleic acid levels and low saturated fat levels with soybean germplasm that contains mutations in soybean genes that confer low linolenic acid phenotypes. Soybean plants and seeds produced by these methods are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/080,087, filed Apr. 5, 2011 and incorporated herein by reference inits entirety, which is a continuation of U.S. patent application Ser.No. 12/713,388, now U.S. Pat. No. 7,943,818, filed Feb. 26, 2010 andincorporated herein by reference in its entirety, which is a division ofU.S. patent application Ser. No. 11/684,413, now U.S. Pat. No.7,790,953, filed Mar. 9, 2007, which claims the benefit of U.S.Provisional Patent Application No. 60/781,519, filed Mar. 10, 2006, bothof which are herein incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

APPENDIX

Not Applicable.

INCORPORATION OF SEQUENCE LISTING

A text file of the Sequence Listing contained in the file named“95832_Seq_listing_ST25.txt” which is 74,492 bytes (measured inMS-Windows®) in size and which was created on Apr. 4, 2011, iselectronically filed herewith and is incorporated by reference in itsentirety. This Sequence Listing consists of SEQ ID NO:1-65.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to methods of making soybean plantsthat produce soybean seed with altered oil compositions and, moreparticularly, to methods where soybean seed with a mid oleic, lowlinolenic phenotype or soybean seed with a mid oleic, low saturate, lowlinolenic phenotype are produced.

2. Related Art

Plant oils are used in a variety of applications. Novel vegetable oilcompositions and improved approaches to obtain oil compositions, frombiosynthetic or natural plant sources, are needed. Depending upon theintended oil use, various different fatty acid compositions are desired.Plants, especially species which synthesize large amounts of oils inseeds, are an important source of oils both for edible and industrialuses. Seed oils are composed almost entirely of triacylglycerols inwhich fatty acids are esterified to the three hydroxyl groups ofglycerol.

Soybean oil typically contains about 16-20% saturated fatty acids:13-16% palmitate and 3-4% stearate. See generally Gunstone et al., TheLipid Handbook, Chapman & Hall, London (1994). Soybean oils have beenmodified by various breeding methods to create benefits for specificmarkets. However, a soybean oil that is broadly beneficial to majorsoybean oil users such as consumers of salad oil, cooking oil and fryingoil, and industrial markets such as biodiesel and biolube markets, isnot available. Prior soybean oils were either too expensive or lacked animportant food quality property such as oxidative stability, good friedfood flavor or saturated fat content, or an important biodiesel propertysuch as appropriate nitric oxide emissions or cold tolerance or coldflow.

Higher plants synthesize fatty acids via a common metabolic pathway—thefatty acid synthetase (FAS) pathway, which is located in the plastids.β-ketoacyl-ACP synthases are important rate-limiting enzymes in the FASof plant cells and exist in several versions. β-ketoacyl-ACP synthase Icatalyzes chain elongation to palmitoyl-ACP (C16:0), whereasβ-ketoacyl-ACP synthase II catalyzes chain elongation to stearoyl-ACP(C18:0). β-ketoacyl-ACP synthase IV is a variant of β-ketoacyl-ACPsynthase II, and can also catalyze chain elongation to 18:0-ACP. Insoybean, the major products of FAS are 16:0-ACP and 18:0-ACP. Thedesaturation of 18:0-ACP to form 18:1-ACP is catalyzed by aplastid-localized soluble delta-9 desaturase (also referred to as“stearoyl-ACP desaturase”). See Voelker et al., 52 Annu. Rev. PlantPhysiol. Plant Mol. Biol. 335-61 (2001).

The products of the plastidial FAS and delta-9 desaturase, 16:0-ACP,18:0-ACP, and 18:1-ACP, are hydrolyzed by specific thioesterases (FAT).Plant thioesterases can be classified into two gene families based onsequence homology and substrate preference. The first family, FATA,includes long chain acyl-ACP thioesterases having activity primarily on18:1-ACP. Enzymes of the second family, FATB, commonly utilize 16:0-ACP(palmitoyl-ACP), 18:0-ACP (stearoyl-ACP), and 18:1-ACP (oleoyl-ACP).Such thioesterases have an important role in determining chain lengthduring de novo fatty acid biosynthesis in plants, and thus these enzymesare useful in the provision of various modifications of fatty acylcompositions, particularly with respect to the relative proportions ofvarious fatty acyl groups that are present in seed storage oils.

The products of the FATA and FATB reactions, the free fatty acids, leavethe plastids and are converted to their respective acyl-CoA esters.Acyl-CoAs are substrates for the lipid-biosynthesis pathway (KennedyPathway), which is located in the endoplasmic reticulum (ER). Thispathway is responsible for membrane lipid formation as well as thebiosynthesis of triacylglycerols, which constitute the seed oil. In theER there are additional membrane-bound desaturases, which can furtherdesaturate 18:1 to polyunsaturated fatty acids. A delta-12 desaturase(FAD2) catalyzes the insertion of a double bond into 18:1 (oleic acid),forming linoleic acid (18:2). A delta-15 desaturase (FAD3) catalyzes theinsertion of a double bond into 18:2, forming linolenic acid (18:3).

Inhibition of the endogenous FAD2 gene through use of transgenes thatinhibit the expression of FAD2 has been shown to confer a desirablemid-oleic acid (18:1) phenotype (i.e. soybean seed comprising about 50%and 75% oleic acid by weight). Transgenes and transgenic plants thatprovide for inhibition of the endogenous FAD2 gene expression and amid-oleic phenotype are disclosed in U.S. Pat. No. 7,067,722. Incontrast, wild type soybean plants that lack FAD2 inhibiting transgenestypically produce seed with oleic acid compositions of less than 20%.

Soybean oil typically contains about 8% of linolenic acid (18:3) thatresults in reduced stability and flavor. The levels of linolenic acid(18:3) in soybean oil can be reduced by hydrogenation to improve bothstability and flavor (Dutton et al., 1951; Lui and White, 1992).Unfortunately, hydrogenation results in the production of trans fattyacids, which increases the risk for coronary heart disease when consumed(Hu et al., 1997).

Conventional breeding has also been used to generate soybean lines withthe linolenic levels ranging from 1%-6% (Ross et al. Crop Science,40:383; 2000; Wilson et al. J. Oleo Sci., 50:5, 87, 2001; Wilson Lipidtechnology September 1999). Varieties of low linolenic acid soybean havebeen produced through mutation, screening and breeding (Fehr et al.,1992; Rahman and Takagi, 1997; Ross et al., 2000; Byrum et al., 1997;Stoisin et al., 1998). Certain soybean varieties with a linolenic acidcontent of about 1% or lower have been obtained (U.S. Pat. Nos.5,534,425 and 5,714,670). More recently, methods for obtaining soybeanplants with both low levels of linolenic acid levels as well as theyield and growth characteristics of agronomically elite soybeanvarieties have been disclosed (U.S. Patent Application 2006/0107348).

Oleic acid has one double bond, but is still relatively stable at hightemperatures, and oils with high levels of oleic acid are suitable forcooking and other processes where heating is required. Recently,increased consumption of high oleic oils has been recommended, becauseoleic acid appears to lower blood levels of low density lipoproteins(“LDLs”) without affecting levels of high density lipoproteins (“HDLs”).However, some limitation of oleic acid levels is desirable, because whenoleic acid is degraded at high temperatures, it creates negative flavorcompounds and diminishes the positive flavors created by the oxidationof linoleic acid. Neff et al., JAOCS, 77:1303-1313 (2000); Warner etal., J. Agric. Food Chem. 49:899-905 (2001). It is thus preferable touse oils with oleic acid levels that are 65-85% or less by weight, inorder to limit off-flavors in food applications such as frying oil andfried food. Other preferred oils have oleic acid levels that are greaterthan 55% by weight in order to improve oxidative stability.

For many oil applications, saturated fatty acid levels of less than 8%by weight or even less than about 2-3% by weight are desirable.Saturated fatty acids have high melting points which are undesirable inmany applications. When used as a feedstock or fuel, saturated fattyacids cause clouding at low temperatures, and confer poor cold flowproperties such as pour points and cold filter plugging points to thefuel. Oil products containing low saturated fatty acid levels may bepreferred by consumers and the food industry because they are perceivedas healthier and/or may be labeled as “saturated fat free” in accordancewith FDA guidelines. In addition, low saturate oils reduce or eliminatethe need to winterize the oil for food applications such as salad oils.In biodiesel and lubricant applications oils with low saturated fattyacid levels confer improved cold flow properties and do not cloud at lowtemperatures.

Soybean lines that produce seed with mid-oleic, low-linoleic acidcontent would be very desirable. Unfortunately, attempts to combine themid oleic and low linolenic traits via genetic engineering approacheshave been problematic. Transgenic lines where both the delta-12desaturase (FAD2) and the delta-15 desaturase (FAD3) genes have beensuppressed have seed with low linolenic levels, but the oleic acidlevels are typically above the range defined for mid oleic. However, themethods disclosed here enable production of low linolenic soybean seedsthat also have oleic acid levels in the mid oleic range of 55-80%.Furthermore, these methods do not entail hydrogenation processes andthus avoid the production of undesirable trans-fats.

Soybean lines that produce seed with mid-oleic, low saturate,low-linoleic acid content would be also very desirable. Methodsdisclosed here enable production of low linolenic soybean seeds thatalso have oleic acid levels in the mid oleic range of 55-80% andsaturated fatty acid levels of less than 8%.

SUMMARY OF THE INVENTION

It is in view of the above problems that the present invention wasdeveloped. The invention first relates to a method of producing asoybean plant comprising a linolenic acid content of less than about 6%of total seed fatty acids by weight and an oleic acid content of about55% to about 80% of total seed fatty acids by weight. This method of theinvention is practiced by a first step of making one or more soybeanplants that comprise a transgene that decreases the expression of anendogenous soybean FAD2-1 gene and at least one loss-of-functionmutation in an endogenous soybean FAD3 gene, a second step of obtainingat least one seed from said soybean plant obtained from the first step,a third step of determining a percentage of the total seed fatty acidcontent by weight of linolenic acid and oleic acid for the seed from thesecond step, and then identifying a soybean plant that yields seedhaving a seed fatty acid composition comprising a linolenic acid contentof less than about 6% of total seed fatty acids by weight and an oleicacid content of about 55% to about 80% of total seed fatty acids byweight.

In other embodiments of this method, the soybean plants that are made inthe first step comprise at least two loss of function mutations in atleast two endogenous soybean FAD3 genes. These loss of functionmutations can be located in the endogenous soybean FAD3-1B and FAD3-1Cgenes. In this embodiment of the method, the soybean plants identifiedin the third step of the method comprise a linolenic acid content ofless than about 3% of total seed fatty acids by weight and an oleic acidcontent of about 55% to about 80% of total seed fatty acids by weight.

In certain embodiments of this method, the transgene can furthercomprise a transgene that confers herbicide tolerance. The herbicidetolerance transgene may confer tolerance to glyphosate. In specificembodiments of the invention, the transgene comprises sequences locatedbetween the T-DNA border sequences of pMON68504, pCGN5469, pCGN5471, orpCGN5485 that are integrated into a chromosome of said plant.

In the third step of the method, the percentage of the total seed fattyacid content by weight of linolenic acid and oleic acid is determined bya lipid analysis technique. This lipid analysis technique comprises oneor more techniques selected from the group consisting of gaschromatography/flame ionization detection, gas chromatography/massspectroscopy, thin layer chromatography/flame ionization detection,liquid chromatography/mass spectrometry, liquidchromatography/electrospray ionization-mass spectrometry and liquidchromatography/electrospray ionization-tandem mass spectroscopy.

The soybean plant comprising a transgene that decreases the expressionof an endogenous soybean FAD2-1 gene and at least one loss-of-functionmutation in an endogenous soybean FAD3 gene can be made by crossing afirst soybean parent line comprising the transgene with a second soybeanparent line comprising at least one loss-of-function mutation in anendogenous soybean FAD3 gene to obtain an F1 soybean plant that isheterozygous for the transgene and heterozygous for at least one loss offunction mutation in a FAD3 gene and then selfing F1 progeny plants fromthe cross to obtain an F2 soybean plant that is homozygous for saidtransgene and homozygous for at least one loss of function mutation in aFAD3 gene. In certain embodiments of this method, the second soybeanparent line comprises at least two loss of function mutations in atleast two endogenous soybean FAD3 genes. The two endogenous soybean FAD3genes can be FAD3-1B and FAD3-1C. In this method, the F1 soybean plantthat is heterozygous for the transgene and for at least one loss offunction mutation in a FAD3 gene is obtained in step (i) by subjecting aplurality of F1 plants to at least one DNA analysis technique permittingidentification of an F1 plant that is heterozygous for said transgeneand for at least one loss of function mutation in a FAD3 gene.Similarly, the F2 soybean plant that is homozygous for the transgene andhomozygous for at least one loss of function mutation in a FAD3 gene isobtained in step (ii) by subjecting a plurality of F2 plants to at leastone DNA analysis technique permitting identification of an F2 plant thatis homozygous for said transgene and homozygous for at least one loss offunction mutation in a FAD3 gene. The DNA analysis technique comprisesone or more techniques selected from the group consisting of PCRanalysis, quantitative PCR analysis, SNP analysis, AFLP analysis, RFLPanalysis and RAPD analysis. In certain embodiments of this invention,the DNA analysis technique comprises detection of at least one singlenucleotide polymorphism at a position in the FAD3-1C gene sequencecorresponding to nucleotide 687, 1129, 1203, 2316, 3292, 3360 or 3743 ofSEQ ID NO:62, detection of a deletion in the FAD3-1C gene of SEQ IDNO:62, or detection of at least one single nucleotide polymorphism in asoybean FAD3-1C promoter sequence corresponding to a guanine atnucleotide 334, a cytosine at nucleotide 364, a thymine at nucleotide385, an adenine at nucleotide 387, a cytosine at nucleotide 393, aguanine at nucleotide 729 and a cytosine at nucleotide 747 of SEQ IDNO:63. In other embodiments of this invention, the DNA analysistechnique comprises detection of a single nucleotide polymorphism in asoybean FAD3-1B gene comprising a substitution of a thymine residue fora cytosine residue at a position in the FAD3-1 b gene sequencecorresponding to nucleotide 2021 of SEQ ID NO:61. In this method, thetransgene can further comprise a transgene that confers herbicidetolerance and the F1 soybean plant that is heterozygous for saidtransgene is obtained in step (i) by subjecting a plurality of F1 plantsto herbicide selection for said transgene. Similarly, when the transgenefurther comprises a transgene that confers herbicide tolerance, aplurality of F2 plants enriched for F2 soybean plants that arehomozygous for said transgene are obtained in step (ii) by subjectingsaid plurality of F2 plants to herbicide selection for said transgene.This method can also further comprise the step iii) of selfing the F2progeny plant that are homozygous for the transgene and homozygous forat least one loss of function mutation in a FAD3 gene from step (ii) toobtain an F3 soybean plant.

An alternative method of making soybean plants that comprise a transgenethat decreases the expression of an endogenous soybean FAD2-1 gene andat least one loss-of-function mutation in an endogenous soybean FAD3gene involves direct transformation of soybean plants or cellscomprising the mutation with the transgene. Thus this soybean plant ismade in the first step of the invention by transforming a soybean plantor plant cell comprising at least one loss-of-function mutation in anendogenous soybean FAD3 gene with a transgene that decreases theexpression of endogenous soybean FAD2-1 gene to obtain an R0 soybeanplant with least one loss of function mutation in a FAD3 gene that isheterozygous for said transgene, selfing the R0 progeny plant from theprevious step to obtain an R1 soybean plant that is homozygous for thetransgene and homozygous for at least one loss of function mutation in aFAD3 gene, thereby obtaining a soybean plant comprising a transgene thatdecreases the expression of an endogenous soybean FAD2-1 gene and atleast one loss-of-function mutation in an endogenous soybean FAD3 gene.In certain embodiments of this method, the transgene further comprisessequences that confer a herbicide tolerance trait. In other embodimentsof the invention, the transgene further comprises sequences that conferglyphosate tolerance.

This invention also encompasses soybean plants produced by theaforementioned methods of the invention as well as plant parts ofsoybean plants produced by the methods of the invention. The soybeanplant part produced can be pollen, an ovule, a meristem, a leaf, a stem,a root, or a cell. Progeny soybean plants from the soybean plantsproduced by these methods are also contemplated by this invention. Theinvention also encompasses seed of the soybean plant produced by themethods of the invention, where this seed has a fatty acid compositioncomprising a linolenic acid content of less than about 6% of total seedfatty acids by weight and an oleic acid content of about 55% to about80% of total seed fatty acids by weight. The invention furtherencompasses seed of the soybean plant produced by methods whereinsoybean plants comprising at least two loss of function mutations in atleast two endogenous soybean FAD3 genes are used, said seed having afatty acid composition comprising a linolenic acid content of less thanabout 3% of total seed fatty acids by weight and an oleic acid contentof about 55% to about 80% of total seed fatty acids by weight.

This invention also provides a method of obtaining a soybean plant withan altered seed oil fatty acid composition comprising the steps of: a)crossing a first soybean parent line having a seed oil fatty acidcomposition comprising a linolenic acid content of less than about 3% oftotal fatty acids by weight with a second soybean parent line having aseed oil fatty acid composition wherein the content of at least onefatty acid other than linoleic acid is altered by at least 50% whencompared to the corresponding fatty acid content of a commodity soybeanoil, said second soybean parent line comprising a transgene that altersthe content of at least one fatty acid other than linoleic acid; and b)obtaining a progeny plant exhibiting a seed oil fatty acid compositioncomprising a linolenic acid content of less than 3% of total fatty acidsby weight and a content of at least one fatty acid other than linoleicacid that is altered by at least 50% when compared to the correspondingfatty acid content of a commodity soybean oil, thereby obtaining asoybean plant with an altered seed oil fatty acid composition. In thismethod, the fatty acid other than linolenic acid is selected from thegroup consisting of lauric acid, myristic acid, palmitic acid, stearicacid, stearidonic acid, oleic acid, linoleic acid, γ-linoleic acid,eicosapentaenoic acid and docosahexaenoic acid.

The invention also relates to a method of producing a soybean plantcomprising a linolenic acid content of less than about 6% of total seedfatty acids by weight, a saturated fatty acid content of less than about8% by weight and an oleic acid content of about 55% to about 80% oftotal seed fatty acids by weight. This method of the invention ispracticed by a first step of making one or more soybean plants thatcomprise at least one transgene that decreases the expression of both anendogenous soybean FAD2-1 and an endogenous FATB gene, and at least oneloss-of-function mutation in an endogenous soybean FAD3 gene, a secondstep of obtaining at least one seed from said soybean plant obtainedfrom the first step, a third step of determining a percentage of thetotal seed fatty acid content by weight of linolenic acid, saturatedfatty acids and oleic acid for the seed from the second step, and thenidentifying a soybean plant that yields seed having a seed fatty acidcomposition comprising a linolenic acid content of less than about 6% oftotal seed fatty acids by weight, a saturated fatty acid content of lessthan about 8% by weight and an oleic acid content of about 55% to about80% of total seed fatty acids by weight.

In other embodiments of this method, the soybean plants that are made inthe first step comprise at least two loss of function mutations in atleast two endogenous soybean FAD3 genes. These loss of functionmutations can be located in the endogenous soybean FAD3-1B and FAD3-1Cgenes. In this embodiment of the method, the soybean plants identifiedin the third step of the method can comprise a linolenic acid content ofless than about 3% of total seed fatty acids by weight, a saturatedfatty acid content of less than about 8% by weight and an oleic acidcontent of about 55% to about 80% of total seed fatty acids by weight.

In certain embodiments of this method, the transgene can furthercomprise a transgene that confers herbicide tolerance. The transgene canconfer tolerance to glyphosate. The transgene that confers resistance toglyphosate can encode a CP4 EPSPS gene.

In the third step of the method, the percentage of the total seed fattyacid content by weight of linolenic acid, saturated fatty acids andoleic acid is determined by a lipid analysis technique. This lipidanalysis technique comprises one or more techniques selected from thegroup consisting of gas chromatography/flame ionization detection, gaschromatography/mass spectroscopy, thin layer chromatography/flameionization detection, liquid chromatography/mass spectrometry, liquidchromatography/electrospray ionization-mass spectrometry and liquidchromatography/electrospray ionization-tandem mass spectroscopy.

The soybean plant comprising at least one transgene that decreases theexpression of both an endogenous soybean FAD2-1 and an endogenous FATBgene and at least one loss-of-function mutation in an endogenous soybeanFAD3 gene can be made by crossing a first soybean parent line comprisingthe transgene with a second soybean parent line comprising at least oneloss-of-function mutation in an endogenous soybean FAD3 gene to obtainan F1 soybean plant that is heterozygous for the transgene(s) andheterozygous for at least one loss of function mutation in a FAD3 geneand then selfing F1 progeny plants from the cross to obtain an F2soybean plant that is homozygous for said transgene and homozygous forat least one loss of function mutation in a FAD3 gene. In certainembodiments of this method, the second soybean parent line comprises atleast two loss of function mutations in at least two endogenous soybeanFAD3 genes. The two endogenous soybean FAD3 genes can be FAD3-1B andFAD3-1C. In this method, the F1 soybean plant that is heterozygous forthe transgene and for at least one loss of function mutation in a FAD3gene is obtained in step (i) by subjecting a plurality of F1 plants toat least one DNA analysis technique permitting identification of an F1plant that is heterozygous for said transgene and for at least one lossof function mutation in a FAD3 gene. Similarly, the F2 soybean plantthat is homozygous for the transgene and homozygous for at least oneloss of function mutation in a FAD3 gene is obtained in step (ii) bysubjecting a plurality of F2 plants to at least one DNA analysistechnique permitting identification of an F2 plant that is homozygousfor said transgene and homozygous for at least one loss of functionmutation in a FAD3 gene. The DNA analysis technique comprises one ormore techniques selected from the group consisting of PCR analysis,quantitative PCR analysis, SNP analysis, AFLP analysis, RFLP analysisand RAPD analysis. In certain embodiments of this invention, the DNAanalysis technique comprises detection of at least one single nucleotidepolymorphism at a position in the FAD3-1C gene sequence corresponding tonucleotide 687, 1129, 1203, 2316, 3292, 3360 or 3743 of SEQ ID NO:62,detection of a deletion in the FAD3-1C gene of SEQ ID NO:62, ordetection of at least one single nucleotide polymorphism in a soybeanFAD3-1C promoter sequence corresponding to a guanine at nucleotide 334,a cytosine at nucleotide 364, a thymine at nucleotide 385, an adenine atnucleotide 387, a cytosine at nucleotide 393, a guanine at nucleotide729 and a cytosine at nucleotide 747 of SEQ ID NO:63. In otherembodiments of this invention, the DNA analysis technique comprisesdetection of single nucleotide polymorphism in a soybean FAD3-1B genecomprising a substitution of a thymine residue for a cytosine residue ata position in the Fad3-1b gene sequence corresponding to nucleotide 2021of SEQ ID NO:61. In this method, the transgene can further comprise atransgene that confers herbicide tolerance and the F1 soybean plant thatis heterozygous for said transgene is obtained in step (i) by subjectinga plurality of F1 plants to herbicide selection for said transgene.Similarly, when the transgene further comprises a transgene that confersherbicide tolerance, a plurality of F2 plants enriched for F2 soybeanplants that are homozygous for said transgene are obtained in step (ii)by subjecting said plurality of F2 plants to herbicide selection forsaid transgene. This method can also further comprise the step iii) ofselfing the F2 progeny plant that are homozygous for the transgene andhomozygous for at least one loss of function mutation in a FAD3 genefrom step (ii) to obtain an F3 soybean plant.

An alternative method of making soybean plants that comprise at leastone transgene that decreases the expression of both an endogenoussoybean FAD2-1 and an endogenous soybean FATB gene and an endogenousFATB gene and at least one loss-of-function mutation in an endogenoussoybean FAD3 gene involves direct transformation of soybean plants orcells comprising the mutation with the transgene(s). Thus this soybeanplant is made in the first step of the invention by transforming asoybean plant or plant cell comprising at least one loss-of-functionmutation in an endogenous soybean FAD3 gene with one or moretransgene(s) that decrease the expression of both an endogenous soybeanFAD2-1 and an endogenous soybean FATB gene to obtain an R0 soybean plantwith least one loss of function mutation in a FAD3 gene that isheterozygous for said transgene, selfing the R0 progeny plant from theprevious step to obtain an R1 soybean plant that is homozygous for thetransgene and homozygous for at least one loss of function mutation in aFAD3 gene, thereby obtaining a soybean plant comprising a transgene thatdecreases the expression of an endogenous soybean FAD2-1 gene and atleast one loss-of-function mutation in an endogenous soybean FAD3 gene.In certain embodiments of this method, the transgene further comprisessequences that confer a herbicide tolerance trait. In other embodimentsof the invention, the transgene further comprises sequences that conferglyphosate tolerance.

This invention also encompasses soybean plants produced by theaforementioned methods of the invention as well as plant parts ofsoybean plants produced by the methods of the invention. The soybeanplant part produced can be pollen, an ovule, a meristem, a leaf, a stem,a root, or a cell. Progeny soybean plants from the soybean plantsproduced by these methods are also contemplated by this invention. Theinvention also encompasses seed of the soybean plant produced by themethods of the invention, where this seed has a fatty acid compositioncomprising a linolenic acid content of less than about 6% of total seedfatty acids by weight, a saturated fatty acid content of less than 8% byweight and an oleic acid content of about 55% to about 80% of total seedfatty acids by weight. The invention further encompasses seed of thesoybean plant produced by methods wherein soybean plants comprising atleast two loss of function mutations in at least two endogenous soybeanFAD3 genes are used, said seed having a fatty acid compositioncomprising a linolenic acid content of less than about 3% of total seedfatty acids by weight, a saturated fatty acid content of less than 8% byweight and an oleic acid content of about 55% to about 80% of total seedfatty acids by weight.

Further features and advantages of the present invention, as well as thestructure and operation of various embodiments of the present invention,are described in detail below with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiments of the present inventionand together with the description, serve to explain the principles ofthe invention, In the drawings:

FIG. 1 illustrates pCGN5469, a plant vector for decreasing expression ofthe soybean FAD2-1 gene;

FIG. 2 illustrates pCGN5471, a plant vector for decreasing expression ofthe soybean FAD2-1 gene;

FIG. 3 illustrates pCGN5485, a plant vector for decreasing expression ofthe soybean FAD2-1 gene; and

FIG. 4 illustrates exemplary plant vector configurations for decreasingexpression of one or more genes by using the DNA sequence elements fromthe soybean genes listed in Table 1.

FIG. 5 illustrates exemplary plant vector configurations for decreasingexpression of one or more genes by using the DNA sequence elements fromthe soybean FAD2-1 and/or soybean FATB genes listed in Table 2.

FIG. 6 illustrates exemplary plant vectors for decreasing expression ofboth the endogenous soybean FAD2-1 and FATB genes.

DETAILED DESCRIPTION OF THE INVENTION

Description of the Nucleic Acid Sequences

SEQ ID NO: 1 is a nucleic acid sequence of a FAD2-1A intron 1.

SEQ ID NO: 2 is a nucleic acid sequence of a FAD2-1B intron 1.

SEQ ID NO: 3 is a nucleic acid sequence of a FAD2-1B promoter.

SEQ ID NO: 4 is a nucleic acid sequence of a FAD2-1A genomic clone.

SEQ ID NOs: 5 & 6 are nucleic acid sequences of a FAD2-1A 3′ UTR and5′UTR, respectively.

SEQ ID NOs: 7-13 are nucleic acid sequences of FAD3-1A introns 1, 2, 3A,4, 5, 3B, and 3C, respectively.

SEQ ID NO: 14 is a nucleic acid sequence of a FAD3-1C intron 4.

SEQ ID NO: 15 is a nucleic acid sequence of a partial FAD3-1A genomicclone.

SEQ ID NOs: 16 & 17 are nucleic acid sequences of a FAD3-1A 3′UTR and5′UTR, respectively.

SEQ ID NO: 18 is a nucleic acid sequence of a partial FAD3-1B genomicclone.

SEQ ID NOs: 19-25 are nucleic acid sequences of FAD3-1B introns 1, 2,3A, 3B, 3C, 4, and 5, respectively.

SEQ ID NOs: 26 & 27 are nucleic acid sequences of a FAD3-1B 3′UTR and5′UTR, respectively.

SEQ ID NO: 28 is a nucleic acid sequence of a FATB-1 genomic clone.

SEQ ID NO: 29-35 are nucleic acid sequences of FATB-1 introns I, II,III, IV, V, VI, and VII, respectively.

SEQ ID NOs: 36 & 37 are nucleic acid sequences of a FATB-1 3′UTR and5′UTR, respectively.

SEQ ID NO: 38 is a nucleic acid sequence of a Cuphea pulcherrima KAS Igene.

SEQ ID NO: 39 is a nucleic acid sequence of a Cuphea pulcherrima KAS IVgene.

SEQ ID NOs: 40 & 41 are nucleic acid sequences of Ricinus communis andSimmondsia chinensis delta-9 desaturase genes, respectively.

SEQ ID NO: 42 is a nucleic acid sequence of a FATB-2 cDNA.

SEQ ID NO: 43 is a nucleic acid sequence of a FATB-2 genomic clone.

SEQ ID NOs: 44-47 are nucleic acid sequences of FATB-2 introns I, II,III, and IV respectively.

SEQ ID NOs: 48-60 are nucleic acid sequences of PCR primers.

SEQ ID NO:61 is a FAD3-1B gene sequence that corresponds to SEQ ID NO:1from U.S. patent application Ser. No. 10/176,149.

SEQ ID NO: 62 is a FAD3-1C gene sequence that corresponds to SEQ ID NO:2from U.S. patent application Ser. No. 10/176,149.

SEQ ID NO:63 is a FAD3-1C promoter sequence that corresponds to SEQ IDNO:3 from U.S. patent application Ser. No. 10/176,149.

DEFINITIONS

“ACP” refers to an acyl carrier protein moiety. “Altered seed oilcomposition” refers to a seed oil composition from a transgenic ortransformed plant of the invention which has altered or modified levelsof the fatty acids therein, relative to a seed oil from a plant having asimilar genetic background but that has not been transformed. “Antisensesuppression” refers to gene-specific silencing that is induced by theintroduction of an antisense RNA molecule.

“Agronomically elite”, as used herein, means a genotype that has aculmination of many distinguishable traits such as emergence, vigor,vegetative vigor, disease resistance, seed set, standability andthreshability which allows a producer to harvest a product of commercialsignificance.

“Allele” as used herein, refers to any of one or more alternative formsof a gene locus, all of which alleles relate to a trait orcharacteristic. In a diploid cell or organism, the two alleles of agiven gene occupy corresponding loci on a pair of homologouschromosomes.

“Backcrossing” as used herein, refers to a process in which a breederrepeatedly crosses hybrid progeny, for example a first generation hybrid(F1), back to one of the parents of the hybrid progeny. Backcrossing canbe used to introduce one or more single locus conversions from onegenetic background into another.

“Coexpression of more than one agent such as an mRNA or protein” refersto the simultaneous expression of an agent in overlapping time framesand in the same cell or tissue as another agent. “Coordinated expressionof more than one agent” refers to the coexpression of more than oneagent when the production of transcripts and proteins from such agentsis carried out utilizing a shared or identical promoter.

“Complement” of a nucleic acid sequence refers to the complement of thesequence along its complete length.

“Cosuppression” is the reduction in expression levels, usually at thelevel of RNA, of a particular endogenous gene or gene family by theexpression of a homologous sense construct that is capable oftranscribing mRNA of the same strandedness as the transcript of theendogenous gene. Napoli et al., Plant Cell 2:279-289 (1990); van derKrol et al., Plant Cell 2:291-299 (1990).

A “CP4 EPSPS” or “CP4 5-enolpyruvylshikimate-3-phosphate synthase” geneencodes an enzyme (CP4 EPSPS) capable of conferring a substantial degreeof glyphosate resistance upon the plant cell and plants generatedtherefrom. The CP4 EPSPS sequence may be a CP4 EPSPS sequence derivedfrom Agrobacterium tumefaciens sp. CP4 or a variant or synthetic formthereof, as described in U.S. Pat. No. 5,633,435. Representative CP4EPSPS sequences include, without limitation, those set forth in U.S.Pat. Nos. 5,627,061 and 5,633,435.

“Crossing”, as used herein, refers to the mating of two parent plants.

“Cross-pollination”, as used herein, refers to fertilization by theunion of two gametes from different plants.

“F₁” or “F1”, as used herein, refers to first generation progeny of thecross of two plants.

“F₁ Hybrid” or F1 Hybrid”, as used herein, refers to first generationprogeny of the cross of two non-isogenic plants.

“F₂” or “F2”, as used herein, refers to second generation progeny of thecross of two plants.

“F₃” or “F3”, as used herein, refers to third generation progeny of thecross of two plants.

“Crude soybean oil” refers to soybean oil that has been extracted fromsoybean seeds, but has not been refined, processed, or blended, althoughit may be degummed.

“CTP” refers to a chloroplastic transit peptide, encoded by the“chloroplastic transit peptide coding sequence”.

When referring to proteins and nucleic acids herein, “derived” refers toeither directly (for example, by looking at the sequence of a knownprotein or nucleic acid and preparing a protein or nucleic acid having asequence similar, at least in part, to the sequence of the known proteinor nucleic acid) or indirectly (for example, by obtaining a protein ornucleic acid from an organism which is related to a known protein ornucleic acid) obtaining a protein or nucleic acid from a known proteinor nucleic acid. Other methods of “deriving” a protein or nucleic acidfrom a known protein or nucleic acid are known to one of skill in theart.

Double-stranded RNA (“dsRNA”), double-stranded RNA interference(“dsRNAi”) and RNA interference (“RNAi”) all refer to gene-specificsilencing that is induced by the introduction of a construct capable oftranscribing an at least partially double-stranded RNA molecule. A“dsRNA molecule” and an “RNAi molecule” both refer to a region of an RNAmolecule containing segments with complementary nucleotide sequences andtherefore can hybridize with each other and form double-stranded RNA.Such double-stranded RNA molecules are capable, when introduced into acell or organism, of at least partially reducing the level of an mRNAspecies present in a cell or a cell of an organism. In addition, thedsRNA can be created after assembly in vivo of appropriate DNA fragmentsthrough illegitimate recombination and site-specific recombination asdescribed in International Application No. PCT/US2005/004681, filed onFeb. 11, 2005, which is hereby incorporated by reference in itsentirety.

“Exon” refers to the normal sense of the term as meaning a segment ofnucleic acid molecules, usually DNA, that encodes part of or all of anexpressed protein.

“FAD2” refers to a gene or encoded protein capable of catalyzing theinsertion of a double bond into a fatty acyl moiety at the twelfthposition counted from the carboxyl terminus. FAD2 proteins are alsoreferred to as “Δ12 desaturase” or “omega-6 desaturase”. The term“FAD2-1” is used to refer to a FAD2 gene or protein that is naturallyexpressed in a specific manner in seed tissue, and the term “FAD2-2” isused to refer a FAD2 gene or protein that is (a) a different gene from aFAD2-1 gene or protein and (b) is naturally expressed in multipletissues, including the seed. Representative FAD2 sequences include,without limitation, those set forth in U.S. patent application Ser. No.10/176,149 filed on Jun. 21, 2002, and in SEQ ID NOs: 1-6.

A “FAD3”, “Δ15 desaturase” or “omega-3 desaturase” gene encodes anenzyme (FAD3) capable of catalyzing the insertion of a double bond intoa fatty acyl moiety at the fifteenth position counted from the carboxylterminus. The terms “FAD3-1, FAD3-A, FAD3-B and FAD3-C” are used torefer to FAD3 gene family members that are naturally expressed inmultiple tissues, including the seed. Representative FAD3 sequencesinclude, without limitation, those set forth in U.S. patent applicationSer. No. 10/176,149 filed on Jun. 21, 2002, and in SEQ ID NOs: 7-27.

A “FATB” or “palmitoyl-ACP thioesterase” refers to a gene that encodesan enzyme (FATB) capable of catalyzing the hydrolytic cleavage of thecarbon-sulfur thioester bond in the panthothene prosthetic group ofpalmitoyl-ACP as its preferred reaction. Hydrolysis of other fattyacid-ACP thioesters may also be catalyzed by this enzyme. RepresentativeFATB-1 sequences include, without limitation, those set forth in U.S.Provisional Application No. 60/390,185 filed on Jun. 21, 2002; U.S. Pat.Nos. 5,955,329; 5,723,761; 5,955,650; and 6,331,664; and SEQ ID NOs:28-37. Representative FATB-2 sequences include, without limitation,those set forth in SEQ ID NOs: 42-47.

“Fatty acid” refers to free fatty acids and fatty acyl groups.

“Gene” refers to a nucleic acid sequence that encompasses a 5′ promoterregion associated with the expression of the gene product, any intronand exon regions and 3′ or 5′ untranslated regions associated with theexpression of the gene product.

“Gene silencing” refers to the suppression of gene expression ordown-regulation of gene expression.

A “gene family” is two or more genes in an organism which encodeproteins that exhibit similar functional attributes, and a “gene familymember” is any gene of the gene family found within the genetic materialof the plant, e.g., a “FAD2 gene family member” is any FAD2 gene foundwithin the genetic material of the plant. An example of two members of agene family are FAD2-1 and FAD2-2. A gene family can be additionallyclassified by the similarity of the nucleic acid sequences. A gene,FAD2, for example, includes alleles at that locus. Preferably, a genefamily member exhibits at least 60%, more preferably at least 70%, morepreferably at least 80% nucleic acid sequence identity in the codingsequence portion of the gene.

“Genotype”, as used herein, refers to the genetic constitution of a cellor organism.

As used herein, “Heterologous” means not naturally occurring together.

A nucleic acid molecule is said to be “introduced” if it is insertedinto a cell or organism as a result of human manipulation, no matter howindirect. Examples of introduced nucleic acid molecules include, but arenot limited to, nucleic acids that have been introduced into cells viatransformation, transfection, injection, and projection, and those thathave been introduced into an organism via methods including, but notlimited to, conjugation, endocytosis, and phagocytosis.

“Intron” refers to the normal sense of the term as meaning a segment ofnucleic acid molecules, usually DNA, that does not encode part of or allof an expressed protein, and which, in endogenous conditions, istranscribed into RNA molecules, but which is spliced out of theendogenous RNA before the RNA is translated into a protein. An “introndsRNA molecule” and an “intron RNAi molecule” both refer to adouble-stranded RNA molecule capable, when introduced into a cell ororganism, of at least partially reducing the level of an mRNA speciespresent in a cell or a cell of an organism where the double-stranded RNAmolecule exhibits sufficient identity to an intron of a gene present inthe cell or organism to reduce the level of an mRNA containing thatintron sequence.

A “low saturate” soybean seed oil composition contains between 3.6 and 8percent saturated fatty acids by weight.

A “low linolenic” oil composition contains less than about 3% linolenicacid by weight of the total fatty acids by weight.

A “mid-oleic soybean seed” is a seed having between 55% and 85% oleicacid present in the oil composition of the seed by weight.

The term “non-coding” refers to sequences of nucleic acid molecules thatdo not encode part or all of an expressed protein. Non-coding sequencesinclude but are not limited to introns, promoter regions, 3′untranslated regions (3′UTRs), and 5′ untranslated regions (5′UTRs).

The term “oil composition” refers to levels of fatty acids.

“Phenotype”, as used herein, refers to the detectable characteristics ofa cell or organism, which characteristics are the manifestation of geneexpression.

A promoter that is “operably linked” to one or more nucleic acidsequences is capable of driving expression of one or more nucleic acidsequences, including multiple coding or non-coding nucleic acidsequences arranged in a polycistronic configuration.

“Physically linked” nucleic acid sequences are nucleic acid sequencesthat are found on a single nucleic acid molecule. A “plant” includesreference to whole plants, plant organs (e.g., leaves, stems, roots,etc.), seeds, and plant cells and progeny of the same. The term “plantcell” includes, without limitation, seed suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, and microspores. “Plant promoters,”include, without limitation, plant viral promoters, promoters derivedfrom plants, and synthetic promoters capable of functioning in a plantcell to promote the expression of an mRNA.

A “polycistronic gene” or “polycistronic mRNA” is any gene or mRNA thatcontains transcribed nucleic acid sequences which correspond to nucleicacid sequences of more than one gene targeted for suppression. It isunderstood that such polycistronic genes or mRNAs may contain sequencesthat correspond to introns, 5′UTRs, 3′UTRs, transit peptide encodingsequences, exons, or combinations thereof, and that a recombinantpolycistronic gene or mRNA might, for example without limitation,contain sequences that correspond to one or more UTRs from one gene andone or more introns from a second gene.

As used herein, the term “R₀”, “R0”, “R₀ generation” or “R0 generation”refers to a transformed plant obtained by regeneration of a transformedplant cell.

As used herein, the term “R₁” “R1”, “R₁ generation” or “R1 generation”refers to seeds obtained from a selfed transgenic R₀ plant. R₁ plantsare grown from the R₁ seeds.

A “seed-specific promoter” refers to a promoter that is activepreferentially or exclusively in a seed. “Preferential activity” refersto promoter activity that is substantially greater in the seed than inother tissues, organs or organelles of the plant. “Seed-specific”includes without limitation activity in the aleurone layer, endosperm,and/or embryo of the seed.

“Sense intron suppression” refers to gene silencing that is induced bythe introduction of a sense intron or fragment thereof. Sense intronsuppression is described, for example by Fillatti in PCT WO 01/14538 A2.

“Simultaneous expression” of more than one agent such as an mRNA orprotein refers to the expression of an agent at the same time as anotheragent. Such expression may only overlap in part and may also occur indifferent tissue or at different levels.

“Total oil level” refers to the total aggregate amount of fatty acidwithout regard to the type of fatty acid. As used herein, total oillevel does not include the glycerol backbone.

“Transgene” refers to a nucleic acid sequence associated with theexpression of a gene introduced into an organism. A transgene includes,but is not limited to, a gene endogenous or a gene not naturallyoccurring in the organism.

A “transgenic plant” is any plant that stably incorporates a transgenein a manner that facilitates transmission of that transgene from a plantby any sexual or asexual method.

A “zero saturate” soybean seed oil composition contains less than 3.6percent saturated fatty acids by weight.

A “loss-of-function mutation” is a mutation in the coding sequence of agene, which causes the function of the gene product, usually a protein,to be either reduced or completely absent. A loss-of-function mutationcan, for instance, be caused by the truncation of the gene productbecause of a frameshift or nonsense mutation. A phenotype associatedwith an allele with a loss of function mutation can be either recessiveor dominant.

A cell or organism can have a family of more than one gene encoding aparticular enzyme, and the capital letter that follows the geneterminology (A, B, C) is used to designate the family member, i.e.,FAD2-1A is a different gene family member from FAD2-1B. Similarly,FAD3-1A, FAD3-1B, and FAD3-1C represent distinct members of the FAD3-1gene family. Loss of function alleles of various genes are representedin lowercase followed by a minus sign (i.e. fad3-1b- andfad3-1c-represent loss of function alleles of the FAD3-1B and FAD3-1Cgenes, respectively).

As used herein, any range set forth is inclusive of the end points ofthe range unless otherwise stated.

A. Transgenes that Decrease the Expression of the Endogenous SoybeanFAD2-1 Gene

Various transgenes that decrease the expression of the endogenoussoybean FAD2-1 gene can be used to practice the methods of theinvention. By suppressing, at least partially reducing, reducing,substantially reducing, or effectively eliminating the expression of theendogenous FAD2 gene, the amount of FAD2 protein available in a plantcell is decreased, i.e. the steady-state levels of the FAD2 protein arereduced. Thus, a decrease in expression of FAD2 protein in the soybeancell can result in an increased proportion of mono-unsaturated fattyacids such as oleate (C18:1). Soybean plants that contain transgenesthat decrease the expression of the endogenous soybean FAD2-1 andproduce seed with increased oleic acid are described in U.S. Pat. No.7,067,722.

Various transgenes that decrease the expression of both an endogenoussoybean FAD2-1 and an endogenous soybean FATB gene can be used topractice the methods of the invention for production of soybean plantswith a low linolenic, low saturate, mid-oleic acid phenotype. Bysuppressing, at least partially reducing, reducing, substantiallyreducing, or effectively eliminating the expression of the endogenousFATB gene, the amount of FATB protein available in a plant cell isdecreased, i.e. the steady-state levels of the FATB protein are reduced.When the amount of FATB is decreased in a plant cell, a decreased amountof saturated fatty acids such as palmitate and stearate can be provided.Thus, a decrease of FATB can result in an increased proportion ofunsaturated fatty acids such as oleate (18:1).

Various methods for decreasing expression of either: 1) the endogenoussoybean FAD2-1 gene(s) or 2) both the endogenous soybean FAD2-1 and FATBgene(s) in soybean plants and seed are contemplated by this invention,including, but not limited to, antisense suppression, co-suppression,ribozymes, combinations of sense and antisense (double-stranded RNAi),promoter silencing, and use of DNA binding proteins such as zinc fingerproteins. The general practice of these methods with respect to variousendogenous plant genes is described in WO 98/53083, WO 01/14538, andU.S. Pat. No. 5,759,829. Suppression of gene expression in plants, alsoknown as gene silencing, occurs at both the transcriptional level andpost-transcriptional level. Certain of these gene silencing mechanismsare associated with nucleic acid homology at the DNA or RNA level. Suchhomology refers to similarity in DNA or protein sequences within thesame species or among different species. Gene silencing occurs if theDNA sequence introduced to a host cell is sufficiently homologous to anendogenous gene that transcription of the introduced DNA sequence willinduce transcriptional or post transcriptional gene silencing of theendogenous gene. To practice this invention, DNA sequences with at least50%, about 60%, or about 70% identical over the entire length of a DNAsequence of a soybean FAD2-1 or FATB coding region or non-coding region,or to a nucleic acid sequence that is complementary to a soybean FAD2-1or FATB coding or non-coding region, have sufficient homology forsuppression of steady state expression levels of FAD2-1 or FATB whenintroduced into soybean plants as transgenes. The transgenes of theinvention more preferably comprise DNA sequences that are, over theirentire length, at least 80% identical; at least 85% identical; at least90% identical; at least 95% identical; at least 97% identical; at least98% identical; at least 99% identical; or 100% identical to a soybeanFAD2-1 or FATB gene coding region or non-coding region, or to a nucleicacid sequence that is complementary to a soybean FAD2-1 or FATB genecoding or non-coding region. The DNA sequences with the above indicatedlevels of identity to the soybean FAD2-1 or FAT gene(s) may be codingsequences, intron sequences, 3′UTR sequences, 5′UTR sequences, promotersequences, other non-coding sequences, or any combination of theforegoing. The intron may be located between exons, or located within a5′ or 3′ UTR of a plant gene. The coding sequence is preferably afraction of a protein encoding frame that does not encode a protein withFAD2 enzymatic activity. However, it is recognized that in certaininstances, such as in cosuppression, DNA sequences that encode anenzymatically active FAD2 or FATB protein can be used to decreaseexpression of the endogenous soybean FAD2-1 or FATB gene(s).

It is also understood that DNA sequences with the above indicated levelsof identity to the soybean FAD2-1 gene that are useful in the methods ofthis invention can be derived from any soybean FAD2 gene, the soybeanFAD2-1A gene (SEQ ID NO:4), the soybean FAD2-1A intron (SEQ ID NO:1),Soybean FAD2-1B introns (SEQ ID NO:2 or SEQ ID NO:3), the soybean FAD-2gene, alleles of the soybean FAD2-1 gene, alleles of the soybean FAD2-2gene, and from FAD2 genes derived from other leguminous plants such asMedicago sp., Pisum sp., Vicia sp., Phaseolus sp., and Pisum sp. It isthus clear that the DNA sequence with the indicated levels of identityto the soybean FAD2-1 sequence can be derived from multiple sources. DNAsequences with the indicated levels of sequence identity can also beobtained synthetically.

Similarly, it is also understood that DNA sequences with the aboveindicated levels of identity to the soybean FATB gene that are useful inthe methods of this invention can be derived from any soybean FATB gene,a soybean FATB-1 gene (SEQ ID NO:28), soybean FATB-1 introns (SEQ IDNO:29-35), soybean FATB-1 5′UTR (SEQ ID NO:36), soybean FATB-1 3′UTR(SEQ ID NO:37), the soybean FATB-2 gene (SEQ ID NO:43), alleles of thesoybean FATB-1, alleles of the soybean FATB-2 gene, and from FATB genesderived from other leguminous plants such as Medicago sp., Pisum sp.,Vicia sp., Phaseolus sp., and Pisum sp. It is thus clear that the DNAsequence with the indicated levels of identity to the soybean FAD2-1sequence can be derived from multiple sources. DNA sequences with theindicated levels of sequence identity can also be obtainedsynthetically.

In the methods of this invention, transgenes specifically designed toproduce double-stranded RNA (dsRNA) molecules with homology to theFAD2-1 gene can also induce FAD2-1 sequence-specific silencing and beused to decrease expression of the endogenous soybean FAD2-1 gene. Thesense strand sequences of the dsRNA can be separated from the antisensesequences by a spacer sequence, preferably one that promotes theformation of a dsRNA molecule. Examples of such spacer sequences includethose set forth in Wesley et al., Plant J., 27(6):581-90 (2001), andHamilton et al., Plant J., 15:737-746 (1988). In a preferred aspect, thespacer sequence is capable of forming a hairpin structure as illustratedin Wesley et al., supra. Particularly preferred spacer sequences in thiscontext are plant introns or parts thereof. A particularly preferredplant intron is a spliceable intron. Spliceable introns include, but arenot limited to, an intron selected from the group consisting of PDKintron, FADS-1A or FAD3-1B intron #5, FAD3 intron #1, FAD3 intron #3A,FAD3 intron #3B, FAD3 intron #3C, FAD3 intron #4, FAD3 intron #5, FAD2intron #1, and FAD2-2 intron. The sense-oriented, non-coding moleculesmay be, optionally separated from the corresponding antisense-orientedmolecules by a spacer segment of DNA. The spacer segment can be a genefragment or artificial DNA. The spacer segment can be short tofacilitate forming hairpin dsRNA or long to facilitate dsRNA without ahairpin structure. The spacer can be provided by extending the length ofone of the sense or antisense molecules as disclosed in US 2005/0176670A1. Alternatively, a right-border-right-border (“RB-RB”) sequence can becreated after insertion into the plant genome as disclosed in U.S.Patent Application 2005/0183170.

The transgenes of the invention will typically include a promoterfunctional in a plant cell, or a plant promoter, that is operably linkedto an aforementioned DNA sequence that decreases expression of anendogenous soybean FAD2-1 or FATB gene. Design of such a vector isgenerally within the skill of the art (See, e.g., Plant MolecularBiology: A Laboratory Manual, Clark (ed.), Springer, N.Y. (1997)).However, it is recognized that constructs or vectors may also contain apromoterless gene that may utilize an endogenous promoter uponinsertion. A number of promoters that are active in plant cells havebeen described in the literature such as the CaMV 35S and FMV promoters.Enhanced or duplicated versions of the CaMV 35S and FMV 35S promoterscan also be used to express an aforementioned DNA sequence thatdecreases expression of an endogenous FAD2-1 gene (Odell et al., Nature313: 810-812 (1985); U.S. Pat. No. 5,378,619). Additional promoters thatmay be utilized are described, for example, in U.S. Pat. Nos. 5,378,619;5,391,725; 5,428,147; 5,447,858; 5,608,144; 5,608,144; 5,614,399;5,633,441; 5,633,435; and 4,633,436. In addition, a tissue specificenhancer can be used with a basal plant promoter. Basal promoterstypically comprise a “TATA” box and an mRNA cap site but lack enhancerelements required for high levels of expression.

Particularly preferred promoters for use in the transgenes of theinstant invention are promoters that express a DNA sequence thatdecreases expression of an endogenous soybean FAD2-1 or FATB gene inseeds or fruits. Indeed, in a preferred embodiment, the promoter used isa seed-specific promoter. Examples of such seed-specific promotersinclude the 5′ regulatory regions from such genes as napin (Kridl etal., Seed Sci. Res. 1:209-219 (1991)), phaseolin, stearoyl-ACPdesaturase, 7Sα, 7Sα′ (Chen et al., Proc. Natl. Acad. Sci., 83:8560-8564(1986)), USP, arcelin and oleosin. Preferred promoters for expression inthe seed are 7Sα, 7Sα′, napin, and FAD2-1A promoters.

Constructs or vectors will also typically include a 3′ transcriptionalterminator or 3′ polyadenylation signal that is operably linked to anaforementioned DNA sequence that decreases expression of an endogenoussoybean FAD2-1 or FATB gene. The transcriptional termination signal canbe any transcriptional termination signal functional in a plant, or anyplant transcriptional termination signal. Preferred transcriptionaltermination signals include, but are not limited to, a pea Rubisco E9 3′sequence, a Brassica napin 3′ sequence, a tml 3′ sequence, and anAgrobacterium tumor-inducing (Ti) plasmid nopaline synthase (NOS) gene3′ sequence. It is understood that this group of exemplarypolyadenylation regions is non-limiting and that one skilled in the artcould employ other polyadenylation regions that are not explicitly citedhere in the practice of this invention.

Finally, it is also recognized that transgenes of the invention can beinserted in plant transformation vectors that also comprise genes thatencode selectable or scoreable markers. The selectable marker gene canbe a gene encoding a neomycin phosphotransferase protein, aphosphinothricin acetyltransferase protein, a glyphosate resistant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) protein, ahygromycin phosphotransferase protein, a dihydropteroate synthaseprotein, a sulfonylurea insensitive acetolactate synthase protein, anatrazine insensitive Q protein, a nitrilase protein capable of degradingbromoxynil, a dehalogenase protein capable of degrading dalapon, a2,4-dichlorophenoxyacetate monoxygenase protein, a methotrexateinsensitive dihydrofolate reductase protein, and an aminoethylcysteineinsensitive octopine synthase protein. The corresponding selectiveagents used in conjunction with each gene can be: neomycin (for neomycinphosphotransferase protein selection), phosphinotricin (forphosphinothricin acetyltransferase protein selection), glyphosate (forglyphosate resistant 5-enol-pyruvylshikimate-3-phosphate synthase(EPSPS) protein selection), hygromycin (for hygromycinphosphotransferase protein selection), sulfadiazine (for adihydropteroate synthase protein selection), chlorsulfuron (for asulfonylurea insensitive acetolactate synthase protein selection),atrazine (for an atrazine insensitive Q protein selection), bromoxinyl(for a nitrilase protein selection), dalapon (for a dehalogenase proteinselection), 2,4-dichlorophenoxyacetic acid (for a2,4-dichlorophenoxyacetate monoxygenase protein selection), methotrexate(for a methotrexate insensitive dihydrofolate reductase proteinselection), or aminoethylcysteine (for an aminoethylcysteine insensitiveoctopine synthase protein selection). A preferred selectable marker geneis a CP4 EPSPS gene that confers resistance to the herbicide glyphosate.The scoreable marker gene can be a gene encoding a beta-glucuronidaseprotein, a green fluorescent protein, a yellow fluorescent protein, abeta-galactosidase protein, a luciferase protein derived from a lucgene, a luciferase protein derived from a lux gene, a sialidase protein,streptomycin phosphotransferase protein, a nopaline synthase protein, anoctopine synthase protein or a chloramphenicol acetyl transferaseprotein.

The above-described nucleic acid molecules are embodiments which achievethe objects, features and advantages of the present invention. It is notintended that the present invention be limited to the illustratedembodiments. The arrangement of the sequences in the first and secondsets of DNA sequences within the nucleic acid molecule is not limited tothe illustrated and described arrangements, and may be altered in anymanner suitable for achieving the objects, features and advantages ofthe present invention as described herein and illustrated in theaccompanying drawings.

B. Transgenic Organisms, and Methods for Producing Same

Any of the nucleic acid molecules and constructs of the invention may beintroduced into a soybean plant or plant cell in a permanent ortransient manner. Methods and technology for introduction of DNA intosoybean plant cells are well known to those of skill in the art, andvirtually any method by which nucleic acid molecules may be introducedinto a cell is suitable for use in the present invention. Non-limitingexamples of suitable methods include: chemical methods; physical methodssuch as microinjection, electroporation, the gene gun, microprojectilebombardment, and vacuum infiltration; viral vectors; andreceptor-mediated mechanisms. Other methods of cell transformation canalso be used and include but are not limited to introduction of DNA intoplants by direct DNA transfer into pollen, by direct injection of DNAinto reproductive organs of a plant, or by direct injection of DNA intothe cells of immature embryos followed by the rehydration of desiccatedembryos.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells. See, e.g., Fraley et al.,Bio/Technology 3:629-635 (1985); Rogers et al., Methods Enzymol.153:253-277 (1987). The region of DNA to be transferred is defined bythe border sequences and intervening DNA is usually inserted into theplant genome. Spielmann et al., Mol. Gen. Genet. 205:34 (1986). ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations.Klee et al., In: Plant DNA Infectious Agents, Hohn and Schell (eds.),Springer-Verlag, New York, pp. 179-203 (1985). Agrobacterium-mediatedtransformation of soybean is specifically described in U.S. Pat. No.7,002,058.

Transgenic plants are typically obtained by linking the gene of interest(i.e., in this case a transgene that decreases expression of anendogenous soybean FAD2-1 gene or that decreases expression of both anFAD2-1 gene or FATB gene) to a selectable marker gene, introducing thelinked transgenes into a plant cell, a plant tissue or a plant by anyone of the methods described above, and regenerating or otherwiserecovering the transgenic plant under conditions requiring expression ofsaid selectable marker gene for plant growth. Exemplary selectablemarker genes and the corresponding selective agents have been describedin preceding sections of this description of the invention.

Transgenic plants can also be obtained by linking a gene of interest(i.e. in this case a transgene that decreases expression of anendogenous soybean FAD2-1 gene or that decreases expression of both anFAD2-1 gene or FATB gene) to a scoreable marker gene, introducing thelinked transgenes into a plant cell by any one of the methods describedabove, and regenerating the transgenic plants from transformed plantcells that test positive for expression of the scoreable marker gene.Exemplary scoreable marker genes have been described in precedingsections of this description of the invention.

The regeneration, development and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art. See generally, Maliga et al., Methods in PlantMolecular Biology, Cold Spring Harbor Press (1995); Weissbach andWeissbach, In: Methods for Plant Molecular Biology, Academic Press, SanDiego, Calif. (1988). Plants of the present invention can be part of orgenerated from a breeding program, and may also be reproduced usingapomixis. Methods for the production of apomictic plants are known inthe art. See, e.g., U.S. Pat. No. 5,811,636.

A particular method of obtaining low linolenic/mid-oleic soybean plantscontemplated herein entails the direct transformation of soybeanvarieties comprising at least one loss-of-function mutation in anendogenous soybean FAD3 gene with a transgene that decreases theexpression of an endogenous soybean FAD2-1 gene. Examples of soybeanvarieties comprising at least one loss-of-function mutation in anendogenous soybean FAD3 gene include A5, C1640, 6P248, N98-44, T27111,T27190, T26767, T26830, and Soyola™ soybean (see U.S. Patent Application20060107348 now U.S. Pat. No. 7,442,850 and Burton et al., Crop Sci.44:687-688, 2004). It is also contemplated that other soybean lines thatcomprise at least one loss-of-function mutation in an endogenous soybeanFAD3 gene and that possess agronomically elite growth and/or yieldcharacteristics produced by the marker-assisted breeding methodsdisclosed in U.S. Patent Application 20060107348, now U.S. Pat. No.7,442,850 could be directly transformed with a transgene that decreasesthe expression of an endogenous soybean FAD2-1 gene. Alternatively, itis also contemplated that soybean lines that comprise at least oneloss-of-function mutation in an endogenous soybean FAD3 gene and thatare amenable to transformation can be produced by the marker-assistedbreeding methods disclosed in U.S. patent application Ser. No.11/239,676, now U.S. Pat. No. 7,442,850. Soybean plants comprising atleast one loss-of-function mutation in an endogenous soybean FAD3 geneand that are amenable to transformation can also be directly transformedwith a transgene that decreases the expression of an endogenous soybeanFAD2-1 gene. Three other low linolenic soybeans that could be directlytransformed by the methods of the invention include BARC12, which is adeterminant maturity group #3 line, Vistive™ soybean lines (Monsanto,St. Louis, Mo., USA), or 0137648/01AHKW-38, which is a yellow hilum L2NUL line.

It is also contemplated that the low linolenic soybean plants that aredirectly transformed with the transgene in the methods of the inventioncan be derived from soybean germplasm comprising soybean plant genomicregions that contain fad3-1b-, fad3-1c-, or both fad3-1b- andfad3-1c-alleles that confer decreased linolenic acid content. Suchsingle nucleotide polymorphisms associated with the low linolenicsoybean phenotype are described in U.S. patent application Ser. No.11/239,676, now U.S. Pat. No. 7,442,850. In certain embodiments, asoybean genomic region that confers the low linolenic acid contentphenotype is characterized by a single nucleotide polymorphism at aposition in the FAD3-1B gene sequence corresponding to nucleotide 2021of SEQ ID NO:61. In another embodiment, the soybean genomic region thatconfers the low linolenic acid content phenotype is characterized by asingle nucleotide polymorphism at a position in the FAD3-1C genesequence corresponding to nucleotide 687, 1129, 1203, 2316, 3292, 3360or 3743 of SEQ ID NO:62. In another embodiment, the soybean genomicregion that confers the low linolenic acid content phenotype ischaracterized by a single nucleotide polymorphism at a position in theFAD3-1C promoter corresponding to nucleotide 334, 364, 385, 387, 393,729 or 747 of SEQ ID NO:63. In another embodiment, the soybean genomicregion that confers the low linolenic acid content phenotype ischaracterized by a deletion in the FAD3-1C gene. The soybean genomicregions that confer the low linolenic acid content phenotype can also becharacterized by both a single nucleotide polymorphism at a position inthe FAD3-1B gene sequence corresponding to nucleotide 2021 of SEQ IDNO:61 and a deletion in the FAD3-1c gene sequence. The soybean genomicregions that confer the low linolenic acid content phenotype can also becharacterized by both a single nucleotide polymorphism at a position inthe FAD3-1B gene sequence corresponding to nucleotide 2021 of SEQ IDNO:61 and a polymorphism in the FAD3-1C promoter, such as a singlenucleotide polymorphism at a position corresponding to nucleotide 334,364, 385, 387, 393, 729 or 747 of SEQ ID NO:63. Soybean germplasmcomprising a deletion in the Soybean FAD3-1C gene is useful in thepractice of these methods and can be obtained from soybean lines thatinclude but are not limited to soybean lines 6P248, T27111, T27190,T26767, T26830 and A5, described in U.S. patent application Ser. No.11/239,676, now U.S. Pat. No. 7,442,850.

Detecting the single nucleotide polymorphisms may be carried out by anymethod, including PCR, single strand conformational polymorphismanalysis, denaturing gradient gel electrophoresis, cleavage fragmentlength polymorphism analysis and/or DNA sequencing as described in U.S.patent application Ser. No. 11/239,676, now U.S. Pat. No. 7,442,850.Alternatively, the single nucleotide polymorphism can be detected by anyone assay selected from the group consisting of single base extension(SBE), allele-specific primer extension sequencing (ASPE), sequencing,universal PCR, allele specific extension, hybridization, massspectrometry, ligation, extension-ligation, and FlapEndonuclease-mediated assays. Primers and methods for detection of theaforementioned FAD3-1B and FAD3-1C genetic polymorphisms are describedin U.S. patent application Ser. No. 11/239,676, now U.S. Pat. No.7,442,850. Deletions such as those in the FAD3-1C gene can be detectedby methods including but not limited to PCR, hybridization, cleavagefragment length polymorphism analysis and/or DNA sequencing-basedmethods.

Direct transformation methods as described above can also be used toobtain low linolenic/low saturate/mid-oleic soybean plants. In thesemethods, the aforementioned low linolenic soybean plants are directlytransformed with transgenes that decrease the expression of both anendogenous soybean FAD2-1 and an endogenous soybean FATB gene forproduction of soybean plants with a low linolenic, low saturate,mid-oleic acid phenotype.

Transgenes that may be used in plant transformation or transfection maybe any of the transgenes that decrease expression of either: 1) theendogenous soybean FAD2-1 gene(s) or 2) both the endogenous soybeanFAD2-1 and FATB gene(s). It is further contemplated that vectorscomprising transgenes that decrease expression of either: 1) theendogenous soybean FAD2-1 gene(s) or 2) both the endogenous soybeanFAD2-1 and FATB gene(s) can also comprise or be genetically combinedwith additional transgenes. For example, additional transgenes thatexpress other genes that affect oil composition, pathogen resistance,yield, morphology, protein composition, amino acid composition, starchcomposition, and phytate level are described in U.S. patent applicationSer. No. 11/239,676, now U.S. Pat. No. 7,442,850 and can be combinedwith the transgenes and low linolenic mutants described herein.

It is not intended that the present invention be limited to theillustrated embodiments. Exemplary nucleic acid molecules have beendescribed in Part A of the Detailed Description, and furthernon-limiting exemplary nucleic acid molecules are described below andillustrated in FIGS. 1-4, and in the Examples.

C. Crosses of Soybean Plants Containing Transgenes

In another aspect, a plant of the invention can be crossed with anotherplant that is transgenic or non-transgenic. A plant can be crossed withanother plant that has an oil composition containing modified levels offatty acids, for example without limitation, a variety with an oilcomposition having a lower level of linolenic acid. In a preferredembodiment, a plant of the present invention is crossed with a varietywith less than 3% by weight linolenic acid. In another embodiment of theinvention, a plant of the present invention is crossed with anotherplant having greater than 20% by weight stearic acid. Such plants havingmodified levels of fatty acids are known in the art and described, forexample, in Hawkins and Kridl (1998) Plant Journal 13(6):743-752 andU.S. Pat. No. 6,365,802.

In particular, crosses of soybean plants comprising a transgene thateither decrease the expression of an endogenous soybean FAD2-1 gene ordecrease the expression of both the endogenous soybean FAD2-1 and FATBgene(s) with soybean varieties comprising at least one loss-of-functionmutation in an endogenous soybean FAD3 gene are contemplated by themethods of this invention. Examples of soybean varieties comprising atleast one loss-of-function mutation in an endogenous soybean FAD3 geneinclude A5, C1640, 6P248, N98-44, T27111, T27190, T26767, T26830, andSoyola™ soybean (see U.S. Patent Application 20060107348, now U.S. Pat.No. 7,442,850 and Burton et al., Crop Sci. 44:687-688, 2004). It is alsocontemplated that other soybean lines that comprise at least oneloss-of-function mutation in an endogenous soybean FAD3 gene and thatpossess agronomically elite growth and/or yield characteristics producedby the marker-assisted breeding methods disclosed in see U.S. PatentApplication 20060107348, now U.S. Pat. No. 7,442,850 could be crossedwith soybean plants comprising a transgene that decreases the expressionof an endogenous soybean FAD2-1 gene. Three other low linolenic crossingparents that could be used in the methods of the invention includeBARC12, which is a determinant maturity group #3 line, Vistive™ soybeanlines, or 0137648/01AHKW-38, which is a yellow hilum L2 NUL line.

It is also contemplated that the low linolenic soybean plants used inthe cross to the transgene(s) can be derived from soybean germplasmcomprising soybean plant genomic regions that contain fad3-1b-,fad3-1c-, or both fad3-1b- and fad3-1c-alleles that confer decreasedlinolenic acid content. Such single nucleotide polymorphisms associatedwith the low linolenic soybean phenotype are described in U.S. patentapplication Ser. No. 11/239,676, now U.S. Pat. No. 7,442,850. In certainembodiments, a soybean genomic region that confers the low linolenicacid content phenotype is characterized by a single nucleotidepolymorphism at a position in the FAD3-1B gene sequence corresponding tonucleotide 2021 of SEQ ID NO:61. In another embodiment, the soybeangenomic region that confers the low linolenic acid content phenotype ischaracterized by a single nucleotide polymorphism at a position in theFAD3-1C gene sequence corresponding to nucleotide 687, 1129, 1203, 2316,3292, 3360 or 3743 of SEQ ID NO:62. In another embodiment, the soybeangenomic region that confers the low linolenic acid content phenotype ischaracterized by a single nucleotide polymorphism at a position in theFAD3-1C promoter corresponding to nucleotide 334, 364, 385, 387, 393,729 or 747 of SEQ ID NO:63. In another embodiment, the soybean genomicregion that confers the low linolenic acid content phenotype ischaracterized by a deletion in the FAD3-1C gene. The soybean genomicregions that confer the low linolenic acid content phenotype can also becharacterized by both a single nucleotide polymorphism at a position inthe FAD3-1B gene sequence corresponding to nucleotide 2021 of SEQ IDNO:61 and a deletion in the FAD3-1C gene sequence. The soybean genomicregions that confer the low linolenic acid content phenotype can also becharacterized by both a single nucleotide polymorphism at a position inthe FAD3-1B gene sequence corresponding to nucleotide 2021 of SEQ IDNO:61 and a polymorphism in the FAD3-1C promoter, such as a singlenucleotide polymorphism at a position corresponding to nucleotide 334,364, 385, 387, 393, 729 or 747 of SEQ ID NO:63. Soybean germplasmcomprising a deletion in the Soybean FAD3-1C gene is useful in thepractice of these methods and can be obtained from soybean lines thatinclude but are not limited to soybean lines 6P248, T27111, T27190,T26767, T26830 and A5, described in U.S. patent application Ser. No.11/239,676, now U.S. Pat. No. 7,442,850. Tables 3 and 4 of the Examplesdescribe the association of specific polymorphisms with specific soybeangermplasm or soybean lines that display low linolenic acid phenotypes.

Without being limited by theory, it is further noted that certainpolymorphisms and deletions in certain FAD3-1C genes are potentiallyresponsible in part for the low linolenic acid phenotypes displayed bysoybean plants that carry these polymorphisms or deletions. The SNP at2021 position in SEQ ID NO:61 is a sense mutation that changes an aminoacid residue from Histidine to Tyrosine. The histidine residue has beenfound to be critical in a number of genes involved with desaturation.This particular SNP found caused a mutation in the motifHis-Val-Ile-His-His (SEQ ID NO:64) to His-Val-Ile-His-Tyr (SEQ ID NO:65)in the low linolenic lines. The motif has been associated with alow-linolenic phenotype and is a likely cause for the reduced linolenicacid phenotype observed in soybeans with this polymorphism.

Detecting the single nucleotide polymorphism may be carried out by anymethod, including PCR, single strand conformational polymorphismanalysis, denaturing gradient gel electrophoresis, cleavage fragmentlength polymorphism analysis and/or DNA sequencing as described in U.S.patent application Ser. No. 11/239,676, now U.S. Pat. No. 7,442,850.Alternatively, the single nucleotide polymorphism can be detected by anyone of an assay selected from the group consisting of single baseextension (SBE), allele-specific primer extension sequencing (ASPE),sequencing, universal PCR, allele specific extension, hybridization,mass spectrometry, ligation, extension-ligation, and FlapEndonuclease-mediated assays. Primers and methods for detection of theaforementioned FAD3-1B and FAD3-1C genetic polymorphisms are describedin U.S. patent application Ser. No. 11/239,676, now U.S. Pat. No.7,442,850. Deletions such as those in the FAD3-1C gene can be detectedby methods including but not limited to PCR, hybridization, cleavagefragment length polymorphism analysis and/or DNA sequencing-basedmethods.

Crossing methods as described above can also be used to obtain lowlinolenic/low saturate/mid-oleic soybean plants. In these methods, theaforementioned low linolenic soybean plants are crossed with soybeanplants comprising transgenes that decrease the expression of both anendogenous soybean FAD2-1 and an endogenous soybean FATB gene forproduction of soybean plants with a low linolenic, low saturate,mid-oleic acid phenotype.

It is further contemplated that the crosses of the transgene(s) to thelow linolenic soybean lines can be facilitated by linkage of aselectable marker that confers resistance to a herbicide. For example,in crosses of soybean plants that are heterozygous for the transgenewith plants that are either homozygous or heterozygous for the allele(s)conferring the low linolenic trait, F1 progeny that are heterozygous forthe transgene can be selected by herbicide treatment. Also, F2 plantsderived from F1 plants that are heterozygous for the transgene can beenriched for F2 soybean plants that are homozygous for said transgene bysubjecting said plurality of F2 plants to herbicide selection for thetransgene. Molecular markers that can distinguish soybean plants thatare either heterozygous or homozygous for the transgene can also be usedto identify soybean plants that are homozygous for the transgeneinsertion.

Soybean plants (Glycine max L.) can be crossed by either natural ormechanical techniques. Natural pollination occurs in soybeans either byself pollination or natural cross pollination, which typically is aidedby pollinating organisms. In either natural or artificial crosses,flowering and flowering time are an important consideration. Soybean isa short-day plant, but there is considerable genetic variation forsensitivity to photoperiod. The critical day length for flowering rangesfrom about 13 h for genotypes adapted to tropical latitudes to 24 h forphotoperiod-insensitive genotypes grown at higher latitudes. Soybeansseem to be insensitive to day length for 9 days after emergence.Photoperiods shorter than the critical day length are required for 7 to26 days to complete flower induction.

Soybean flowers typically are self-pollinated on the day the corollaopens. The stigma is receptive to pollen about 1 day before anthesis andremains receptive for 2 days after anthesis, if the flower petals arenot removed. Filaments of nine stamens are fused, and the one nearestthe standard is free. The stamens form a ring below the stigma untilabout 1 day before anthesis, then their filaments begin to elongaterapidly and elevate the anthers around the stigma. The anthers dehisceon the day of anthesis, pollen grains fall on the stigma, and within 10h the pollen tubes reach the ovary and fertilization is completed.Self-pollination occurs naturally in soybean with no manipulation of theflowers. For the crossing of two soybean plants, it is typicallypreferable, although not required, to utilize artificial hybridization.In artificial hybridization, the flower used as a female in a cross ismanually cross pollinated prior to maturation of pollen from the flower,thereby preventing self fertilization, or alternatively, the male partsof the flower are emasculated using a technique known in the art.Techniques for emasculating the male parts of a soybean flower include,for example, physical removal of the male parts, use of a genetic factorconferring male sterility, and application of a chemical gametocide tothe male parts.

Either with or without emasculation of the female flower, handpollination can be carried out by removing the stamens and pistil with aforceps from a flower of the male parent and gently brushing the anthersagainst the stigma of the female flower. Access to the stamens can beachieved by removing the front sepal and keel petals, or piercing thekeel with closed forceps and allowing them to open to push the petalsaway. Brushing the anthers on the stigma causes them to rupture, and thehighest percentage of successful crosses is obtained when pollen isclearly visible on the stigma. Pollen shed can be checked by tapping theanthers before brushing the stigma. Several male flowers may have to beused to obtain suitable pollen shed when conditions are unfavorable, orthe same male may be used to pollinate several flowers with good pollenshed.

Genetic male sterility is available in soybeans and may be useful tofacilitate hybridization in the context of the current invention,particularly for recurrent selection programs. The distance required forcomplete isolation of a crossing block is not clear; however,out-crossing is less than 0.5% when male-sterile plants are 12 m or morefrom a foreign pollen source (Boerma and Moradshahi, Crop Sci.,15:858-861, 1975). Plants on the boundaries of a crossing block probablysustain the most out-crossing with foreign pollen and can be eliminatedat harvest to minimize contamination.

Once harvested, pods are typically air-dried at not more than 38° C.until the seeds contain 13% moisture or less, then the seeds are removedby hand. Seed can be stored satisfactorily at about 25° C. for up to ayear if relative humidity is 50% or less. In humid climates, germinationpercentage declines rapidly unless the seed is dried to 7% moisture andstored in an air-tight container at room temperature. Long-term storagein any climate is best accomplished by drying seed to 7% moisture andstoring it at 10° C. or less in a room maintained at 50% relativehumidity or in an air-tight container.

F. Products of the Present Invention

The plants of the present invention may be used in whole or in part.Preferred plant parts include reproductive or storage parts. The term“plant parts” as used herein includes, without limitation, seed,endosperm, ovule, pollen, roots, tubers, stems, leaves, stalks, fruit,berries, nuts, bark, pods, seeds and flowers. In a particularlypreferred embodiment of the present invention, the plant part is a seed.

Any of the plants or parts thereof of the present invention may beprocessed to produce a feed, meal, protein, or oil preparation. Aparticularly preferred plant part for this purpose is a seed. In apreferred embodiment the feed, meal, protein or oil preparation isdesigned for livestock animals, fish or humans, or any combination.Methods to produce feed, meal, protein and oil preparations are known inthe art. See, e.g., U.S. Pat. Nos. 4,957,748, 5,100,679, 5,219,596,5,936,069, 6,005,076, 6,146,669, and 6,156,227. In a preferredembodiment, the protein preparation is a high protein preparation. Sucha high protein preparation preferably has a protein content of greaterthan 5% w/v, more preferably 10% w/v, and even more preferably 15% w/v.

In a preferred oil preparation, the oil preparation is a high oilpreparation with an oil content derived from a plant or part thereof ofthe present invention of greater than 5% w/v, more preferably 10% w/v,and even more preferably 15% w/v. In a preferred embodiment the oilpreparation is a liquid and of a volume greater than 1, 5, 10 or 50liters. The present invention provides for oil produced from plants ofthe present invention or generated by a method of the present invention.Such oil may exhibit enhanced oxidative stability. Also, such oil may bea minor or major component of any resultant product.

Moreover, such oil may be blended with other oils. In a preferredembodiment, the oil produced from plants of the present invention orgenerated by a method of the present invention constitutes greater than0.5%, 1%, 5%, 10%, 25%, 50%, 75% or 90% by volume or weight of the oilcomponent of any product. In another embodiment, the oil preparation maybe blended and can constitute greater than 10%, 25%, 35%, 50% or 75% ofthe blend by volume. Oil produced from a plant of the present inventioncan be admixed with one or more organic solvents or petroleumdistillates.

Seeds of the plants may be placed in a container. As used herein, acontainer is any object capable of holding such seeds. A containerpreferably contains greater than about 500, 1,000, 5,000, or 25,000seeds where at least about 10%, 25%, 50%, 75% or 100% of the seeds arederived from a plant of the present invention. The present inventionalso provides a container of over about 10,000, more preferably about20,000, and even more preferably about 40,000 seeds where over about10%, more preferably about 25%, more preferably 50% and even morepreferably about 75% or 90% of the seeds are seeds derived from a plantof the present invention. The present invention also provides acontainer of over about 10 kg, more preferably about 25 kg, and evenmore preferably about 50 kg seeds where over about 10%, more preferablyabout 25%, more preferably about 50% and even more preferably about 75%or 90% of the seeds are seeds derived from a plant of the presentinvention.

Soybean seeds produced by the methods of the invention can comprisevarious oil compositions. An oil produced by soybean seeds produced bythe methods of the invention are referred to below as an “oil of thepresent invention”.

A preferred oil of the present invention has a low saturate oilcomposition, or a zero saturate oil composition. In other preferredembodiments, oils of the present invention have increased oleic acidlevels, reduced saturated fatty acid levels, and reduced polyunsaturatedfatty acid levels. In further preferred embodiments, oils of the presentinvention have increased oleic acid levels and reduced saturated fattyacid levels. In a preferred embodiment, the oil is a soybean oil. Thepercentages of fatty acid content, or fatty acid levels, used hereinrefer to percentages by weight.

In a first embodiment, an oil of the present invention preferably has anoil composition that is 55 to 80% oleic acid, about 12 to 43%polyunsaturates, and 2 to 8% saturated fatty acids; more preferably hasan oil composition that is 55 to 80% oleic acid, about 14 to 42%polyunsaturates, and 3 to 6% saturated fatty acids; and even morepreferably has an oil composition that is 55 to 80% oleic acid, about16.5 to 43% polyunsaturates, and 2 to 3.6% saturated fatty acids.

In a second embodiment, an oil of the present invention preferably hasan oil composition that is 65 to 80% oleic acid, about 12 to 33%polyunsaturates, and 2 to 8% saturated fatty acids; more preferably hasan oil composition that is 65 to 80% oleic acid, about 14 to 32%polyunsaturates, and 3 to 6% saturated fatty acids; and even morepreferably has an oil composition that is 65 to 80% oleic acid, about16.5 to 33% polyunsaturates, and 2 to 3.6% saturated fatty acids.

In a third embodiment, an oil of the present invention preferably has anoil composition that is about 42 to about 85% oleic acid and about 8% toabout 1.5% saturated fatty acids.

In a fourth embodiment, an oil of the present invention has an oilcomposition that is about 42 to about 85% oleic acid, about 8% to about1.5% saturated fatty acids, about 6% to about 15% by weight linolenicacid; more preferably has an oil composition that is about 42 to about85% oleic acid, about 8% to about 1.5% saturated fatty acids, less than35% by weight linolenic acid; and even more preferably has an oilcomposition that is about 42 to about 85% oleic acid, about 8% to about1.5% saturated fatty acids, about 9% by weight linolenic acid.

In a fifth embodiment, an oil of the present invention has an oilcomposition that is about 50% to about 85% oleic acid and about 8% toabout 1.5% saturated fatty acids; more preferably about 50% to about 85%oleic acid, about 8% to about 1.5% saturated fatty acids, about 4% toabout 14% by weight linolenic acid; more preferably has an oilcomposition that is about 50% to about 85% oleic acid, about 8% to about1.5% saturated fatty acids, less than 35% by weight linolenic acid; andeven more preferably has an oil composition that is about 42 to about85% oleic acid, about 8% to about 1.5% saturated fatty acids, about 2%to about 45% by weight linolenic acid.

In another embodiment, an oil of the present invention has an oilcomposition that is about 65-80% oleic acid, about 3-8% saturates, andabout 12-32% polyunsaturates. In another embodiment, an oil of thepresent invention has an oil composition that is about 65-80% oleicacid, about 2-3.5% saturates, and about 16.5-33% polyunsaturates.

In a particularly preferred embodiment, an oil of the present inventionhas an oil composition that is about 47-83% oleic acid and about 5%saturates; about 60-80% oleic acid and about 5% saturates; about 50-85%oleic and about 2-7% saturates; about 55-85% oleic acid and about 2.5-7%saturates; about 47-88% oleic acid and about 3-7% saturates; about43-85% oleic acid and about 5-7% saturates; about 81-85% oleic acid andabout 5% saturates; about 74-83% oleic acid and about 6% saturates;about 65-87% oleic acid and about 6% saturates; about 66-80% oleic acidand about 6% saturates; about 42-77% oleic acid and about 5-8%saturates; about 60-77% oleic acid and about 6% saturates; about 70-81%oleic acid and about 5-7% saturates; about 52-71% oleic acid and about5-7% saturates; about 44-71% oleic acid and about 6% saturates; about61-71% oleic acid and about 8% saturates; about 57-71% oleic acid andabout 7% saturates; about 23-58% oleic acid and about 8-14% saturates;about 20-70% oleic acid and about 6% saturates; about 21-35% oleic acidand about 5-6% saturates; or about 19-28% oleic acid and about 5%saturates.

In other embodiments, the percentage of oleic acid is 50% or greater;55% or greater; 60% or greater; 65% or greater; 70% or greater; 75% orgreater; or 80% or greater; or is a range from 50 to 80%; 55 to 80%; 55to 75%; 55 to 65%; 60 to 85%; 60 to 80%; 60 to 75%; 60 to 70%; 65 to85%; 65 to 80%; 65 to 75%; 65 to 70%; or 69 to 73%. Suitable percentageranges for oleic acid content in oils of the present invention alsoinclude ranges in which the lower limit is selected from the followingpercentages: 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80percent; and the upper limit is selected from the following percentages:60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 percent.

In other embodiments, the percentage of linolenic acid in an oil of thepresent invention is 10% or less; 9% or less; 8% or less; 7% or less; 6%or less; 5% or less; 4.5% or less; 4% or less; 3.5% or less; 3% or less;3.0% or less; 2.5% or less; or 2% or less; or is a range from 0.5 to 2%;0.5 to 3%; 0.5 to 4.5%; 0.5% to 6%; 3 to 5%; 3 to 6%; 3 to 8%; 1 to 2%;1 to 3%; or 1 to 4%.

In these other embodiments, the percentage of saturated fatty acids inan oil composition of the present invention is 15% or less; 14% or less;13% or less; 12% or less, 11% or less; 10% or less; 9% or less; 8% orless; 7% or less; 6% or less; 5% or less; 4% or less; or 3.6% or less;or is a range from 2 to 3%; 2 to 3.6%; 2 to 4%; 2 to 8%; 3 to 15%; 3 to10%; 3 to 8%; 3 to 6%; 3.6 to 7%; 5 to 8%; 7 to 10%; or 10 to 15%.

In other embodiments, saturated fatty acids in an oil of the presentinvention includes the combination of the palmitic and stearic fattyacids. In an embodiment, the percentage of saturated fatty acids rangesfrom about 10% or less; about 9% or less; about 8% or less; about 7% orless; about 6% or less; about 5% or less; about 4.5% or less; about 4%or less; about 3.5% or less; about 3% or less; about 3.0% or less; about2.5% or less; or about 2% or less; or is a range from 0.5 to 2%; 0.5 to3%; 0.5 to 4.5%; 0.5 to 6%; 0.5 to 7%; 0.5 to 8%; 0.5 to 9%; 1 to 4%; 1to 5%; 1 to 6%; 1 to 7%; 1 to 8%; 1 to 9%; 1.5 to 5%; 1.5 to 6%; 1.5 to7%; 1.5 to 8%; 1.5 to 9%; 2 to 5%; 2 to 6%; 2 to 7%; 2 to 8%; 2 to 9%; 3to 5%; 3 to 6%; 3 to 7%; 3 to 8%; 3 to 9%; 4 to 7%; 4 to 8%; 4 to 9%; 5to 7%; 5 to 8%; and 5 to 9%. In these embodiments, suitable percentageranges for saturated fatty acid content in oils of the present inventionalso include ranges in which the lower limit is selected from thefollowing percentages: 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,or 6.5 percent; and the upper limit is selected from the followingpercentages: 11, 10, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or0.5 percent.

G. Modulation of Suppression

Another embodiment of the invention is directed to a method ofmodulating gene suppression levels. Modulation of gene suppression canresult in more or less gene suppression. Suppression of a gene productcan be the result from insertion of a construct of the present inventioninto a plant genome. Similarly, modulation of gene suppression can bethe result from insertion of a construct of the present invention into aplant genome. Other examples of methods to modulate gene suppressioninclude, without limitation, antisense techniques, cosuppression, RNAinterference (dsRNAi), transgenic animals, hybrids, and ribozymes usinga construct of the present invention. The following examples areprovided by way of illustration, and are not intended to be limiting ofthe present invention.

Suppression of a gene can be modulated by altering the length of thetranscribable DNA used for suppression, which sequence is derived fromthe gene targeted for suppression. Many methods can be used forsuppressing a gene using post-transcriptional gene silencing mechanisms.Without being limited to the theory, these methods are believed to havein common the expression of an RNA molecule which hybridizes to anotherRNA molecule. Surprisingly, there can be advantages to using an RNAmolecule of particular lengths to modulate or moderate suppression ofthe steady state expression levels of a targeted endogenous gene.

Gene suppression of FAD2-1 leads to elevated levels of oleic acid andreduction of linoleic acid levels. When FAD2-1 is heavily suppressed,levels of oleic acid can be greater than 65%, which causes a reductionin palmitic acid and linolenic acid levels. For example, when FAD2-1 issuppressed, oleic acid levels can reach 85% and the combined palmiticand stearic acid levels are reduced to about 10%. Similarly,downregulation of FATB results in decreased levels of saturated fattyacids, primarily palmitate. When FAD2 and FATB are suppressed so thatoleic levels are about 85%, saturate levels are about 10%. When FAD2 andFATB are suppressed so that oleic levels are greater than 85%, saturatelevels can fall below 10%.

In light of the present invention, saturate levels can be reduced toless than 10% without enhancing oleic acids above 85%. In oneembodiment, the suppression of FAD2 is modulated by reducing the lengthof FAD2-1 intron introduced into the plant. Less suppression of FAD2results in moderate levels of oleic acid, approximately 40-85% oleicacid. The suppression of FAD2 is reduced as the length of the FAD2-1intron fragment introduced is reduced. For example, a FAD2-1 intronreduced in length by at least 100 contiguous nucleotides can reduce thesuppression of FAD2 and the corresponding increase in oleic acid anddecrease in linoleic acid levels.

The relationship between the decrease in endogenous gene suppression andthe decrease in length of homologous DNA can be determined empiricallyby introducing different lengths of DNA. For example, the amount ofreduction in suppression obtainable by reducing the length of homologousintroduced DNA can be determined by deleting increasing portions of thehomologous DNA being introduced and assaying for expression of thetargeted gene.

Included in the present invention is a method for moderating suppressionof FAD2 while still having a strong reduction of saturate levels in aplant. In such plants, oleic acid levels can range from 40-85%.Similarly, less than full suppression of FATB occurs when the combined3′ and 5′ untranslated regions are introduced as compared to when thefull-length FATB gene is introduced into a host cell. In a like manner,suppression levels of FATB are reduced when the 5′ part of the openreading frame, which mostly encodes the chloroplast transit peptide, isintroduced into a host cell. In cells with FAD2 and FATB suppressedusing methods according to the present invention, oleic acid levels canbe 40-85% while saturate levels can be between 1 to 9 percent.

In one embodiment, the present invention is directed to a method ofmodulating gene suppression to reduce suppression relative to thesuppression from an entire gene element, where an entire gene elementcan be an entire gene, an entire exon, an entire intron, an entiresignal sequence, or an entire UTR, then constructing a recombinantnucleic acid molecule comprising a fragment of the endogenous sequencefrom the gene element; initiating expression of the recombinant nucleicacid molecule in a host cell; and suppressing the endogenous gene withthe recombinant nucleic acid molecule. The gene being suppressed can beany gene, including FAD2 and FATB. In one embodiment, the presentinvention is directed to a method of modulating FAD2 or FATB suppressioncomprising: expressing a partial FAD2 or FATB gene element sequence in ahost cell, where a FAD2 or FATB gene element is from an endogenous FAD2or FATB gene in the host cell and a FAD2 or FATB gene element sequencecan be a FAD2 or FATB gene, a FAD2 or FATB exon, a FAD2 or FATB intron,a FAD2 or FATB transit peptide coding region, or a FAD2 or FATB UTR; andthe partial FAD2 or FATB gene element sequence is less than the entireFAD2 or FATB gene element sequence; and suppressing an endogenous FAD2or FATB with the partial FAD2 or FATB gene element sequence, wheresuppression levels of the FAD2 or FATB endogenous gene in the host cellare less than suppression levels of the FAD2 or FATB endogenous gene ina host cell with a similar genetic background and a second FAD2 or FATBnucleic acid sequence comprising the entire FAD2 or FATB gene elementsequence of the FAD2 or FATB gene element.

In another embodiment, the present invention is directed to a method ofaltering the oil composition of a plant cell by transforming a plantcell with a recombinant nucleic acid molecule which comprises a DNAsequence that suppresses endogenous expression of FAD2, FATB, or FAD2and FATB where the DNA sequence comprises a nucleic acid sequence ofFAD2, FATB, or FAD2 and FATB that is shorter than the entire sequence ofan entire genetic element selected from a gene, an exon, an intron, atransit peptide coding region, a 3′-UTR, a 5′-UTR, and an open readingframe; and growing the plant cell under conditions where transcriptionof said DNA sequence is initiated, whereby the oil composition isaltered relative to a plant cell with a similar genetic background butlacking the recombinant nucleic acid molecule. A gene element of FAD2 orFATB can be shortened in length by 50, 75, 100, 150, 175, 200, 250, 300,350, 400, 450, 500, 600, 800, 1000, 2000, 3000, or 4000 nucleotides. Alength of a gene element of FAD2 or FATB can be 50, 75, 100, 150, 175,200, 220, 250, 300, 320, 350, 400, 420, 450, 500, 550, 600, 800, or 1000nucleotides.

In another embodiment, the present invention is directed to a method ofenhancing oleic acid content and reducing saturated fatty acid contentin a plant seed by: i) shortening the length of an exogenous FAD2 DNAsequence in a host cell until the amount of suppression of FAD2expression from a transformed plant is at least partially reducedrelative to the suppression of FAD2 expression in a host cell with asimilar genetic background and an entire exogenous FAD2 gene DNAsequence; and ii) growing a plant with a nucleic acid moleculecomprising the shortened FAD2 DNA sequence, where the shortened FAD2 DNAsequence at least partially suppresses endogenous expression of FAD2;and iii) cultivating a plant that produces seed with a reduced saturatedfatty acid content relative to seed from a plant having a similargenetic background but lacking the shortened FAD2 DNA sequence. Theamount that the exogenous FAD2 DNA sequence is shortened to at leastpartially reduce suppression of the endogenous FAD2 can be determinedempirically by introducing different lengths of DNA. For example, theamount of reduction in suppression obtainable by reducing the length ofhomologous introduced DNA can be determined by deleting increasingportions of the homologous DNA being introduced and assaying forexpression of the targeted gene. The amount of suppression of FAD2expression can be obtained as an average of three or more, six or more,ten or more, fifteen or more, or twenty or more seeds from a plant.

In another embodiment, the present invention is directed to a method ofproducing a transformed plant having seed with a reduced saturated fattyacid content by transforming a plant cell with a recombinant nucleicacid molecule which comprises a DNA sequence that suppresses theendogenous expression of FAD2 and FATB, where the DNA sequence comprisesa nucleic acid sequence of FAD2 that is shorter than the entire sequenceof an entire genetic element selected from a gene, an exon, an intron, atransit peptide coding region, and a UTR; and growing the transformedplant, where the transformed plant produces seed with a reducedsaturated fatty acid content relative to seed from a plant having asimilar genetic background but lacking said recombinant nucleic acidmolecule.

In another embodiment, the present invention is directed to a method ofmodulating the fatty acid composition of oil from a seed of a temperateoilseed crop by isolating a genetic element of at least 40 nucleotidesin length that is capable of suppressing the expression of an endogenousgene in the fatty acid synthesis pathway; generating more than oneshortened fragment of the genetic element; introducing each of the morethan one shortened fragments into a plant cell of the temperate oilseedcrop to produce transgenic plants; and selecting a transgenic plantcomprising a shortened fragment of determined length and sequence thateffects a desirable change in seed oil fatty acid composition. In apreferred embodiment, the method above also includes constructing arecombinant DNA construct having at least two shortened fragments of twodifferent endogenous genes that effect different desirable changes inseed oil fatty acid composition; introducing the recombinant DNAconstruct into a plant cell of the temperate oilseed crop to producetransgenic plants; and selecting a transgenic plant comprising the atleast two shortened fragments and a fatty acid composition of oil from aseed having more than one desirable change effected by the at least twoshortened fragments.

In another embodiment, the present invention is directed to a soybeanseed exhibiting an oil composition having a strongly reduced saturatedfatty acid content and a moderately enhanced oleic acid content having aDNA sequence that suppresses the endogenous expression of FAD2 in a hostcell, where the DNA sequence has a nucleic acid sequence of FAD2 that isshorter than the entire sequence of an entire genetic element selectedfrom a gene, an exon, an intron, a transit peptide coding region, and aUTR.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Isolation of FATB-2 Sequences

Leaf tissue is obtained from Asgrow soy variety A3244, ground in liquidnitrogen and stored at 80° C. until use. Six ml of SDS Extraction buffer(650 ml sterile ddH20, 100 ml 1M Tris-Cl pH 8, 100 ml 0.25M EDTA, 50 ml20% SDS, 100 ml 5M NaCl, 4 μl beta-mercaptoethanol) is added to 2 ml offrozen/ground leaf tissue, and the mixture is incubated at 65° C. for 45minutes. The sample is shaken every 15 minutes. 2 ml of ice-cold 5Mpotassium acetate is added to the sample, the sample is shaken, and thenis incubated on ice for 20 minutes. 3 ml of CHCl₃ is added to the sampleand the sample is shaken for 10 minutes.

The sample is centrifuged at 10,000 rpm for 20 minutes and thesupernatant is collected. 2 ml of isopropanol is added to thesupernatant and mixed. The sample is then centrifuged at 10,000 rpm for20 minutes and the supernatant is drained. The pellet is resuspended in200 μl RNase and incubated at 65° C. for 20 minutes. 300 μl ammoniumacetate/isopropanol (1:7) is added and mixed. The sample is thencentrifuged at 10,000 rpm for 15 minutes and the supernatant isdiscarded. The pellet is rinsed with 500 μl 80% ethanol and allowed toair dry. The pellet of genomic DNA is then resuspended in 200 μl T10E1(10 mM Tris:1 mM EDTA).

A soy FATB-2 cDNA contig sequence (SEQ ID NO: 42) is used to designthirteen oligonucleotides that span the gene: F1 (SEQ ID NO: 48), F2(SEQ ID NO: 49), F3 (SEQ ID NO: 50), F4 (SEQ ID NO: 51), F5 (SEQ ID NO:52), F6 (SEQ ID NO: 53), F7 (SEQ ID NO: 54), R1 (SEQ ID NO: 55), R2 (SEQID NO: 56), R3 (SEQ ID NO: 57), R4 (SEQ ID NO: 58), R5 (SEQ ID NO: 59),and R6 (SEQ ID NO: 60). The oligonucleotides are used in pairs for PCRamplification from the isolated soy genomic DNA: pair 1 (F1+R1), pair 2(F2+R1), pair 3 (F3+R2), pair 4 (F4+R3), pair 5 (F5+R4), pair 6 (F6+R5),and pair 7 (F7+R6). The PCR amplification for pair 5 is carried out asfollows: 1 cycle, 95° C. for 10 minutes; 30 cycles, 95° C. for 15 sec,43° C. for 30 sec, 72° C. for 45 sec; 1 cycle, 72° C. for 7 minutes. Forall other oligo pairs, PCR amplifications are carried out as follows: 1cycle, 95° C. for 10 minutes; 30 cycles, 95° C. for 15 sec, 48° C. for30 sec, 72° C. for 45 sec; 1 cycle, 72° C. for 7 minutes. Positivefragments are obtained from primer pairs 1, 2, 4, 5, 6 and 7. Eachfragment is cloned into vector pCR2.1 (Invitrogen). Fragments 2, 4, 5and 6 are confirmed and sequenced. These four sequences are aligned toform a genomic sequence for the FATB-2 gene (SEQ ID NO: 43).

Four introns are identified in the soybean FATB-2 gene by comparison ofthe genomic sequence to the cDNA sequence: intron I (SEQ ID NO: 44)spans base 119 to base 1333 of the genomic sequence (SEQ ID NO: 43) andis 1215 bp in length; intron II (SEQ ID NO: 45) spans base 2231 to base2568 of the genomic sequence (SEQ ID NO: 43) and is 338 bp in length;intron III (SEQ ID NO: 46) spans base 2702 to base 3342 of the genomicsequence (SEQ ID NO: 43) and is 641 bp in length; and intron IV (SEQ IDNO: 47) spans base 3457 to base 3823 of the genomic sequence (SEQ ID NO:43) and is 367 bp in length.

Example 2 Suppression Constructs

2A. FAD2-1Constructs

The FAD2-1A intron #1(SEQ ID NO: 1) is cloned into the expressioncassette, pCGN3892, in sense and antisense orientations. The vectorpCGN3892 contains the soybean 7S promoter and a pea rbcS 3′. Both genefusions are then separately ligated into pCGN9372, a vector thatcontains the CP4 EPSPS gene regulated by the FMV promoter. The resultingexpression constructs (pCGN5469 sense and pCGN5471 antisense, depictedin FIGS. 1 and 2, respectively) are used for transformation of soybean.

The FAD2-1B intron (SEQ ID NO: 2) is fused to the 3′ end of the FAD2-1Aintron #1 in plasmid pCGN5468 (contains the soybean 7S promoter fused tothe FAD2-1A intron (sense) and a pea rbcS 3′) or pCGN5470 (contains thesoybean 7S promoter fused to the FAD2-1A intron (antisense) and a pearbcS 3′) in sense and antisense orientation, respectively. The resultingintron combination fusions are then ligated separately into pCGN9372, avector that contains the CP4 EPSPS gene regulated by the FMV promoter.The resulting expression constructs (pCGN5485, FAD2-1A & FAD2-1B intronsense and pCGN5486, FAD2-1A & FAD2-1B intron antisense) are used fortransformation of soybean.

B. FAD3-1Constructs

FAD3-1A introns #1, #2, #4 and #5 (SEQ ID NOs: 7, 8, 10 and 11,respectively), FAD3-1B introns #3C (SEQ ID NO: 23) and #4 (SEQ ID NO:24), are all ligated separately into pCGN3892, in sense or antisenseorientation. pCGN3892 contains the soybean 7S promoter and a pea rbcS3′. These fusions are ligated into pCGN9372, a vector that contains theCP4 EPSPS gene regulated by the FMV promoter for transformation intosoybean. The resulting expression constructs (pCGN5455, FAD3-1A intron#4 sense; pCGN5459, FAD3-1A intron #4 antisense; pCGN5456, FAD3 intron#5 sense; pCGN5460, FAD3-1A intron #5 antisense; pCGN5466, FAD3-1Aintron #2 antisense; pCGN5473, FAD3-1A intron #1 antisense) are used fortransformation of soybean.

2C. FatB Constructs

The soybean FATB-1 intron II sequence (SEQ ID NO: 30) is amplified viaPCR using a FATB-1 partial genomic clone as a template. PCRamplification is carried out as follows: 1 cycle, 95° C. for 10 min; 25cycles, 95° C. for 30 sec, 62° C. for 30 sec, 72° C. for 30 sec; 1cycle, 72° C. for 7 min. PCR amplification results in a product that is854 bp long, including reengineered restriction sites at both ends. ThePCR product is cloned directly into the expression cassette pCGN3892 insense orientation, by way of XhoI sites engineered onto the 5′ ends ofthe PCR primers, to form pMON70674. Vector pCGN3892 contains the soybean7S promoter and a pea rbcS 3′. pMON70674 is then cut with NotI andligated into pMON41164, a vector that contains the CP4 EPSPS generegulated by the FMV promoter. The resulting gene expression construct,pMON70678, is used for transformation of soybean using Agrobacteriummethods.

2D. Combination Constructs

Expression constructs are made containing various permutations of: 1) aFAD2-1 sequences alone (for low linolenic, mid-oleic soybean productionmethods) and 2) combinations of FAD2-1 and FATB DNA sequences. The DNAsequences are any of those described, or illustrated in Table 2, or anyother set of DNA sequences that contain various combinations of sense,antisense, or sense and antisense FAD2 and/or FATB non-coding or codingregions so that they are capable of forming dsRNA constructs, senseco-suppression constructs, antisense constructs, or various combinationsof the foregoing.

FIG. 4 depicts DNA sequences which are capable of expressing senseco-suppression or antisense constructs according to the presentinvention, the non-coding sequences of which are described in Table 1and 2 below. The non-coding sequences may be single sequences,combinations of sequences (e.g., the 5′UTR linked to the 3′UTR), or anycombination of the foregoing. To express a sense co-suppressionconstruct, all of the non-coding sequences are sense sequences, and toexpress an antisense construct, all of the non-coding sequences areantisense sequences. To express sense and antisense constructs, bothsense and antisense non-coding sequences are provided.

FIG. 5 depict several first sets of DNA sequences which are capable ofexpressing dsRNA constructs according to the present invention, thenon-coding sequences of which are described in Tables 1 and 2 below. Thefirst set of DNA sequences depicted in FIG. 5 comprises pairs of relatedsense and antisense sequences, arranged such that, e.g., the RNAexpressed by the first sense sequence is capable of forming adouble-stranded RNA with the antisense RNA expressed by the firstantisense sequence. For example, referring to the topmost vector of FIG.5 and illustrative combination No. 1 (of Table 1), the first set of DNAsequences comprises a sense FAD2-1 sequence, a sense FAD3-1 sequence, anantisense FAD2-1 sequence and an antisense FAD3-1 sequence. Bothantisense sequences correspond to the sense sequences so that theexpression products of the first set of DNA sequences are capable offorming a double-stranded RNA with each other. The sense sequences maybe separated from the antisense sequences by a spacer sequence,preferably one that promotes the formation of a dsRNA molecule. Examplesof such spacer sequences include those set forth in Wesley et al.,supra, and Hamilton et al., Plant J., 15:737-746 (1988). The promoter isany promoter functional in a plant, or any plant promoter. Non-limitingexamples of suitable promoters are described in Part A of the DetailedDescription.

The first set of DNA sequences is inserted in an expression construct ineither the sense or anti-sense orientation using a variety of DNAmanipulation techniques. If convenient restriction sites are present inthe DNA sequences, they are inserted into the expression construct bydigesting with the restriction endonucleases and ligation into theconstruct that has been digested at one or more of the available cloningsites. If convenient restriction sites are not available in the DNAsequences, the DNA of either the construct or the DNA sequences ismodified in a variety of ways to facilitate cloning of the DNA sequencesinto the construct. Examples of methods to modify the DNA include byPCR, synthetic linker or adapter ligation, in vitro site-directedmutagenesis, filling in or cutting back of overhanging 5′ or 3′ ends,and the like. These and other methods of manipulating DNA are well knownto those of ordinary skill in the art.

TABLE 1 Illustrative Non-Coding or Coding Sequences (sense orantisense). Combinations First Second Third Fourth 1 FAD2-1A or BFAD3-1A or B or C 2 FAD3-1A or B or C FAD2-1A or B 3 FAD2-1A or BFAD3-1A or B or C different FAD3-1A or B or C sequence 4 FAD2-1A or BFAD3-1A or B or C FATB-1 5 FAD2-1A or B FATB-1 FAD3-1A or B or C 6FAD3-1A or B or C FAD2-1A or B FATB-1 7 FAD3-1A or B or C FATB-1 FAD2-1Aor B 8 FATB-1 FAD3-1A or B or C FAD2-1A or B 9 FATB-1 FAD2-1A or BFAD3-1A or B or C 10 FAD2-1A or B FAD3-1A or B or C different FAD3-1A orFATB-1 B or C sequence 11 FAD3-1A or B or C FAD2-1A or B differentFAD3-1A or FATB-1 B or C sequence 12 FAD3-1A or B or C different FAD3-1Aor FAD2-1A or B FATB-1 B or C sequence 13 FAD2-1A or B FAD3-1A or B or CFATB-1 different FAD3-1A or B or C sequence 14 FAD3-1A or B or C FAD2-1Aor B FATB-1 different FAD3-1A or B or C sequence 15 FAD3-1A or B or Cdifferent FAD3-1A or FATB-1 FAD2-1A or B B or C sequence 16 FAD2-1A or BFATB-1 FAD3-1A or B or C different FAD3-1A or B or C sequence 17 FAD3-1Aor B or C FATB-1 FAD2-1A or B different FAD3-1A or B or C sequence 18FAD3-1A or B or C FATB-1 different FAD3-1A or FAD2-1A or B B or Csequence 19 FATB-1 FAD2-1A or B FAD3-1A or B or C different FAD3-1A or Bor C sequence 20 FATB-1 FAD3-1A or B or C FAD2-1A or B different FAD3-1Aor B or C sequence 21 FATB-1 FAD3-1A or B or C different FAD3-1A orFAD2-1A or B B or C sequence 22 FAD2-1A or B FAD3-1A or B or C FATB-2 23FAD2-1A or B FATB-2 FAD3-1A or B or C 24 FAD3-1A or B or C FAD2-1A or BFATB-2 25 FAD3-1A or B or C FATB-2 FAD2-1A or B 26 FATB-2 FAD3-1A or Bor C FAD2-1A or B 27 FATB-2 FAD2-1A or B FAD3-1A or B or C 28 FAD2-1A orB FAD3-1A or B or C different FAD3-1A or FATB-2 B or C sequence 29FAD3-1A or B or C FAD2-1A or B different FAD3-1A or FATB-2 B or Csequence 30 FAD3-1A or B or C different FAD3-1A or FAD2-1A or B FATB-2 Bor C sequence 31 FAD2-1A or B FAD3-1A or B or C FATB-2 different FAD3-1Aor B or C sequence 32 FAD3-1A or B or C FAD2-1A or B FATB-2 differentFAD3-1A or B or C sequence 33 FAD3-1A or B or C different FAD3-1A orFATB-2 FAD2-1A or B B or C sequence 34 FAD2-1A or B FATB-2 FAD3-1A or Bor C different FAD3-1A or B or C sequence 35 FAD3-1A or B or C FATB-2FAD2-1A or B different FAD3-1A or B or C sequence 36 FAD3-1A or B or CFATB-2 different FAD3-1A or FAD2-1A or B B or C sequence 37 FATB-2FAD2-1A or B FAD3-1A or B or C different FAD3-1A or B or C sequence 38FATB-2 FAD3-1A or B or C FAD2-1A or B different FAD3-1A or B or Csequence 39 FATB-2 FAD3-1A or B or C different FAD3-1A or FAD2-1A or B Bor C sequence 40 FAD2-1A or B FATB-1 41 FAD2-1A or B FATB-2 42 FAD2-1Aor B FATB-1 FATB-2 43 FAD2-1A FAD2-1B FATB-1 44 FAD2-1A FAD2-1B FATB-1FATB-2 45 FAD2-1A or B FAD2-1A or B 46 FATB-1 or FATB-2 FATB-1 or FATB-2

TABLE 2 Correlation of SEQ ID NOs with Sequences in Table 1 FAD3-FAD2-1A FAD2-1B FAD3-1A FAD3-1B 1C FATB-1 FATB-2 3′UTR SEQ NO: 5 n/a SEQNO: SEQ NO: 26 n/a SEQ NO: n/a 16 36 5′UTR SEQ NO: 6 n/a SEQ NO: SEQ NO:27 n/a SEQ NO: n/a 17 37 5′ + 3′ UTR Linked n/a Linked Linked SEQ n/aLinked SEQ n/a (or 3′ + 5′ SEQ NOs: SEQ NOs: NOs: 26 and NOs: 36 UTR) 5and 6 16 and 17 27 and 37 Intron #1 SEQ NO: 1 SEQ NO: 2 SEQ NO: 7 SEQNO: 19 n/a SEQ NO: SEQ NO: 29 44 Intron #2 n/a n/a SEQ NO: 8 SEQ NO: 20n/a SEQ NO: SEQ NO: 30 45 Intron #3 n/a n/a n/a n/a n/a SEQ NO: SEQ NO:31 46 Intron #3A n/a n/a SEQ NO: 9 SEQ NO: 21 n/a n/a n/a Intron #3B n/an/a SEQ NO: SEQ NO: 22 n/a n/a n/a 12 Intron #3C n/a n/a SEQ NO: SEQ NO:23 n/a n/a n/a 13 Intron #4 n/a n/a SEQ NO: SEQ NO: 24 SEQ SEQ NO: SEQNO: 47 10 NO: 32 14 Intron #5 n/a n/a SEQ NO: SEQ NO: 25 n/a SEQ NO: n/a11 33 Intron #6 n/a n/a n/a n/a n/a SEQ NO: n/a 34 Intron #7 n/a n/a n/an/a n/a SEQ NO: n/a 35

Example 3

3A. Antisense Constructs

Referring now to FIG. 7, soybean FATB-2 non-coding sequences (SEQ IDNOs: 44-47), FATB-1 non-coding sequences (SEQ ID NOs: 29-37), and FAD2-1non-coding sequences (SEQ ID NOs: 1 and 5-6) are amplified via PCR toresult in PCR products that include reengineered restriction sites atboth ends. The PCR products are cloned directly in sense and antisenseorientation into a vector containing the soybean 7Sα′ promoter and a tml3′ termination sequence. The vector is then cut with an appropriaterestriction endonuclease and ligated into pMON80612 a vector thatcontains the CP4 EPSPS gene regulated by the FMV promoter and a peaRubisco E9 3′ termination sequence. The resulting gene expressionconstruct is depicted in the bottom most construct of FIG. 7 and is usedfor transformation using methods as described herein.

3B. In Vivo Assembly

An aspect of the present invention includes a DNA construct thatassembles into a recombinant transcription unit on a plant chromosome inplanta that is capable of forming double-stranded RNA. The assembly ofsuch constructs and the methods for assembling in vivo a recombinanttranscription units for gene suppression are described in InternationalApplication No. PCT/US2005/00681, hereby incorporated by reference inits entirety.

pMON95829 is a construct used for in vivo assembly that has two T-DNAsegments, each flanked by Agrobacterium T-DNA border elements, i.e.right border DNA (RB) and left border DNA (LB). The first T-DNA containsa soybean 7Sα′ promoter operably linking to a soybean FAD2-1A intron 1(SEQ ID NO: 1), which is reduced by 100 contiguous nucleotides from the3′ end and ligated to 42 contiguous nucleotides of a FATB-1a 5′ UTR,followed by the FATB-1A chloroplast transit peptide (“CTP”) codingregion, and a CP4 EPSPS gene operably linking to an enhanced FMVpromoter and a pea Rubisco E9 3′ termination sequence all flanked byAgrobacterium T-DNA border elements, i.e. right border DNA (RB) and leftborder DNA (LB). On the same vector in the second T-DNA segment, flankedby another RB and LB, is a H6 3′ termination sequence operably linkingto a soybean FAD2-1A intron 1 (SEQ ID NO: 1), which is reduced by 100contiguous nucleotides from the 3′ end and ligated to 42 contiguousnucleotides of a FATB-1a 5′ UTR, followed by the FATB-1A chloroplasttransit peptide (“CTP”) coding region. The resulting gene expressionconstruct is used for transformation using methods as described herein.

pMON97595 is a construct used for in vivo assembly that has two T-DNAsegments, each flanked by Agrobacterium T-DNA border elements, i.e.right border DNA (RB) and left border DNA (LB). The first T-DNA segmentcontains a soybean 7Sα′ promoter operably linking to a soybean FAD2-1Aintron 1 (SEQ ID NO: 1), which is reduced by 320 contiguous nucleotidesfrom the 3′ end and ligated to 42 contiguous nucleotides of a FATB-1a 5′UTR followed by the FATB-1a chloroplast transit peptide (“CTP”) codingregion, and a CP4 EPSPS gene operably linking to an enhanced FMVpromoter and a pea rubisco E9 3′ termination sequence, all flanked byAgrobacterium T-DNA border elements, i.e. right border DNA (RB) and leftborder DNA (LB). On the second T-DNA segment, flanked by another RB andLB, is a H6 3′ termination sequence operably linked to a soybean FAD2-1Aintron 1 (SEQ ID NO: 1), which is reduced by 320 contiguous nucleotidesfrom the 3′ end and ligated to 42 contiguous nucleotides of a FATB-1a5′UTR followed by the FATB-1A CTP coding region. The resulting geneexpression construct is used for transformation using methods asdescribed herein.

pMON97581 is a construct used for in vivo assembly that has two T-DNAsegments, each flanked by Agrobacterium T-DNA border elements, i.e.right border DNA (RB) and left border DNA (LB). The first T-DNA segmentcontains a soybean 7Sα′ promoter operably linking to a soybean FAD2-1Aintron 1 (SEQ ID NO: 1), which is reduced by 320 contiguous nucleotidesfrom the 3′ end and ligated to the FATB-1a chloroplast transit peptide(“CTP”) coding region, and a CP4 EPSPS gene operably linking to anenhanced FMV promoter and a pea Rubisco E9 3′ termination sequence, allflanked by Agrobacterium T-DNA border elements, i.e. right border DNA(RB) and left border DNA (LB). On the same construct, in the secondT-DNA segment, flanked by another RB and LB, is a H6 3′ terminationsequence operably linked to a soybean FAD2-1A intron 1 (SEQ ID NO: 1),which is reduced by 320 contiguous nucleotides from the 3′ end andligated to the FATB-1a CTP coding region. The resulting gene expressionconstruct is used for transformation using methods as described herein.

pMON97596 is a construct used for in vivo assembly that has two T-DNAsegments, each flanked by Agrobacterium T-DNA border elements, i.e.right border DNA (RB) and left border DNA (LB). The first T-DNA segmentcontains a soybean 7Sα′ promoter operably linking to a soybean FAD2-1Aintron 1 (SEQ ID NO: 1), which is reduced by 320 contiguous nucleotidesfrom the 3′ end and ligated to the 5′ 180 bp of the FATB-1a chloroplasttransit peptide (“CTP”) coding region, and a CP4 EPSPS gene operablylinking to an enhanced FMV promoter and a pea Rubisco E9 3′ terminationsequence, all flanked by Agrobacterium T-DNA border elements, i.e. rightborder DNA (RB) and left border DNA (LB). On the same construct, in thesecond T-DNA segment, flanked by another RB and LB, is a H6 3′termination sequence operably linked to a soybean FAD2-1A intron 1 (SEQID NO: 1), which is reduced by 320 contiguous nucleotides from the 3′end and ligated to the 5′ 180 bp of the FATB-1a CTP coding region. Theresulting gene expression construct is used for transformation usingmethods as described herein.

pMON97597 is a construct used for in vivo assembly that has two T-DNAsegments, each flanked by Agrobacterium T-DNA border elements, i.e.right border DNA (RB) and left border DNA (LB). The first T-DNA segmentcontains a soybean 7Sα′ promoter operably linking to a soybean FAD2-1Aintron 1 (SEQ ID NO: 1), which is reduced by 320 contiguous nucleotidesfrom the 3′ end and ligated to the 5′ 120 bp of the FATB-1a chloroplasttransit peptide (“CTP”) coding region, and a CP4 EPSPS gene operablylinking to an enhanced FMV promoter and a pea Rubisco E9 3′ terminationsequence, all flanked by Agrobacterium T-DNA border elements, i.e. rightborder DNA (RB) and left border DNA (LB). On the same construct, in thesecond T-DNA segment, flanked by another RB and LB, is a H6 3′termination sequence operably linked to a soybean FAD2-1A intron 1 (SEQID NO: 1), which is reduced by 320 contiguous nucleotides from the 3′end and ligated to the 5′ 120 bp of the FATB-1a CTP coding region. Theresulting gene expression construct is used for transformation usingmethods as described herein.

pMON97598 is a construct used for in vivo assembly that has two T-DNAsegments, each flanked by Agrobacterium T-DNA border elements, i.e.right border DNA (RB) and left border DNA (LB). The first T-DNA segmentcontains a soybean 7Sα′ promoter operably linking to a soybean FAD2-1Aintron 1 (SEQ ID NO: 1), which is reduced by 340 contiguous nucleotidesfrom the 3′ end and ligated to the FATB-1a chloroplast transit peptide(“CTP”) coding region, and a CP4 EPSPS gene operably linking to anenhanced FMV promoter and a pea Rubisco E9 3′ termination sequence, allflanked by Agrobacterium T-DNA border elements, i.e. right border DNA(RB) and left border DNA (LB). On the same construct, in the secondT-DNA segment, flanked by another RB and LB, is a H6 3′ terminationsequence operably linked to a soybean FAD2-1A intron 1 (SEQ ID NO: 1),which is reduced by 340 contiguous nucleotides from the 3′ end andligated to the FATB-1a CTP coding region. The resulting gene expressionconstruct is used for transformation using methods as described herein.

When the two T-DNA segments of the any one of the above constructs (i.e.pMON95829, pMON97595, pMON97581, pMON97597, pMON97598) are inserted intoa single locus of the chromosome of a host organism in a RB to RBorientation, the assembled transcription unit has a soybean 7Sα′promoter operably linking sense and anti-sense-oriented soybean FAD2-1Aintron 1 and FATB-1a DNA fragments. When transcribed, the operablylinked sense and anti-sense oriented RNA sequences are capable offorming double-stranded RNA effective for suppression of FAD2-1A andFATB.

Example 4 Plant Transformation and Analysis

The constructs of Examples 2 and 3 are stably introduced into soybean(for example, Asgrow variety A4922 or Asgrow variety A3244 or Asgrowvariety A3525) by the methods described earlier, including the methodsof McCabe et al., Bio/Technology, 6:923-926 (1988), orAgrobacterium-mediated transformation. Transformed soybean plants areidentified by selection on media containing a selectable agent orherbicide. The herbicide can be glyphosate when a transgene conferringresistance to glyphosate is used. Fatty acid compositions are analyzedfrom seed of soybean lines transformed with the constructs using gaschromatography.

For some applications, modified fatty acid compositions are detected indeveloping seeds, whereas in other instances, such as for analysis ofoil profile, detection of fatty acid modifications occurring later inthe FAS pathway, or for detection of minor modifications to the fattyacid composition, analysis of fatty acid or oil from mature seeds isperformed. Furthermore, analysis of oil and/or fatty acid content ofindividual seeds may be desirable, especially in detection of oilmodification in the segregating R1 seed populations. As used herein, R0generation indicates the plant arising from transformation/regenerationprotocols described herein, the R1 generation indicates seeds grown onthe selfed transgenic R0 plant. R1 plants are grown from the R1 seeds.

Fatty acid compositions are determined for the seed of soybean linestransformed with the constructs of Example 3. One to ten seeds of eachof the transgenic and control soybean lines are ground individuallyusing a tissue homogenizer (Pro Scientific) for oil extraction. Oil fromground soybean seed is extracted overnight in 1.5 ml heptane containingtriheptadecanoin (0.50 mg/ml). Aliquots of 200 μl of the extracted oilare derivatized to methyl esters with the addition of 500 μl sodiummethoxide in absolute methanol. The derivatization reaction is allowedto progress for 20 minutes at 50° C. The reaction is stopped by thesimultaneous addition of 500 μl 10% (w/v) sodium chloride and 400 μlheptane. The resulting fatty acid methyl esters extracted in hexane areresolved by gas chromatography (GC) on a Hewlett-Packard model 6890 GC(Palo Alto, Calif.). The GC was fitted with a Supelcowax 250 column (30m, 0.25 mm id, 0.25 micron film thickness) (Supelco, Bellefonte, Pa.).Column temperature is 175° C. at injection and the temperatureprogrammed from 175° C. to 245° C. to 175° C. at 40° C./min. Injectorand detector temperatures are 250° C. and 270° C., respectively.

Example 5

This example illustrates plant transformation to produce soybean plantswith suppressed genes.

A transformation vector pMON68537 is used to introduce an intron/3′UTRdouble-stranded RNA-forming construct into soybean for suppressing theΔ12 desaturase, Δ15 desaturase, and FATB genes. Vector pMON68537 alsocontains the delta-9 desaturase (FAB2) and the CP4 genes. The pMON68537vector is designed for plant transformation to suppress FAD2, FAD3, andFATB genes and overexpress delta-9 desaturase in soybean. In particular,the construct comprises a 7S alpha promoter operably linked to soybeansense-oriented intron and 3′UTRs, i.e., a FAD2-1A intron #1, a FAD3-1A3′UTR, a FATB-1 3′UTR, a hairpin loop-forming spliceable intron, and acomplementary series of soybean anti-sense-oriented intron and 3′UTR's,i.e., a FATB-1 3′UTR, a FAD3-1A 3′UTR and a FAD2-1A intron #1 and thesoybean FAD2 promoter driving the delta-9 desaturase. The vector isstably introduced into soybean (Asgrow variety A4922) via Agrobacteriumtumefaciens strain ABI (Martinell, U.S. Pat. No. 6,384,301). The CP4selectable marker allows transformed soybean plants to be identified byselection on media containing glyphosate herbicide.

Fatty acid compositions are analyzed from seed of soybean linestransformed with the intron/3′UTR dsRNAi expression constructs using gaschromatography. R1 pooled seed and R1 single seed oil compositionsdemonstrate that the mono- and polyunsaturated fatty acid compositionsare altered in the oil of seeds from transgenic soybean lines ascompared to that of the seed from non-transformed soybean, (See Table3). For instance, FAD2 suppression provides plants with increased amountof oleic acid ester compounds; FAD3 suppression provides plants withdecreased linolenic acid ester compounds; and FATB suppression providesplants with reduced saturated fatty ester compounds, e.g. palmitates andstearates. Selections can be made from such lines depending on thedesired relative fatty acid composition. Fatty acid compositions areanalyzed from seed of soybean lines transformed with constructs usinggas chromatography.

TABLE 3 Fatty acid composition of R1 single seeds from pMON68537 EventsConstruct Event 18:1 18:3 16:0 18:0 18:2 PMON68537 GM_A36305 74.92 4.426.35 2.93 10.24 PMON68537 GM_A36305 74.8 4.33 6.57 2.93 10.23 PMON68537GM_A36305 74.43 3.95 5.98 2.82 11.81 PMON68537 GM_A36305 73.32 3.99 6.793.24 11.48 PMON68537 GM_A36305 72.87 4.33 7.06 3.08 11.7 PMON68537GM_A36305 16.63 9.53 13.5 4.06 55.31 PMON68537 GM_A36305 16.52 9.6113.92 4.24 54.79 PMON68537 GM_A36305 15.67 9.66 13.64 4.19 55.89PMON68537 GM_A36306 77.45 3.93 6.76 2.47 8.4 PMON68537 GM_A36306 74.514.38 6.58 2.47 10.94 PMON68537 GM_A36306 73.21 4.64 7.04 3.08 11.04PMON68537 GM_A36306 72.78 4.4 6.97 2.55 12.21 PMON68537 GM_A36306 71.674.76 6.94 3.25 12.2 PMON68537 GM_A36306 71.01 4.86 7.64 3.05 12.41PMON68537 GM_A36306 69.72 4.76 7.66 2.95 13.75 PMON68537 GM_A36306 17.418.88 13.35 3.85 55.63 PMON68537 GM_A36307 77.22 3.71 6.8 2.77 8.5PMON68537 GM_A36307 76.79 3.65 6.76 2.85 8.75 PMON68537 GM_A36307 71.444.54 7.2 3.58 12.17 PMON68537 GM_A36307 18.83 8.62 13.94 4.02 53.61PMON68537 GM_A36307 18.81 8.38 13.27 3.7 54.97 PMON68537 GM_A36307 15.689.97 14.06 4.55 54.79 PMON68537 GM_A36307 15.28 10.64 14.68 4.43 53.97PMON68537 GM_A36307 14.08 9.36 14.39 4.31 56.89 PMON68537 GM_A3630978.67 3.53 6.09 2.5 8.18 PMON68537 GM_A36309 75.43 3.96 6.7 2.53 10.3PMON68537 GM_A36309 71.41 4.19 6.92 2.74 13.67 PMON68537 GM_A36309 70.514.14 6.85 3.16 14.33 PMON68537 GM_A36309 67.51 5.01 7.45 3.15 15.69PMON68537 GM_A36309 66.99 4.92 7.15 3.9 15.79 PMON68537 GM_A36309 20.098.46 12.41 5 52.97 PMON68537 GM_A36309 15.15 9.73 14.61 3.85 55.79PMON68537 GM_A36310 74.28 4.77 7.31 1.85 10.9 PMON68537 GM_A36310 74.035.43 8.23 1.63 9.66 PMON68537 GM_A36310 73.07 5.09 7.37 1.76 11.75PMON68537 GM_A36310 71.83 5.04 7.78 1.86 12.54 PMON68537 GM_A36310 68.016.26 9.8 1.97 13.13 PMON68537 GM_A36310 67.22 6.28 8.71 3.28 13.45PMON68537 GM_A36310 65.37 6.87 10.01 1.94 14.9 PMON68537 GM_A36310 15.7610.09 13.4 4.28 55.52 PMON68537 GM_A36311 77.87 3.56 5.9 2.46 9.05PMON68537 GM_A36311 75.8 3.87 5.91 2.93 10.22 PMON68537 GM_A36311 75.613.71 6.21 2.56 10.75 PMON68537 GM_A36311 73.68 4.06 6 3.09 11.98PMON68537 GM_A36311 72.66 4.11 6.41 3.14 12.48 PMON68537 GM_A36311 70.894.39 6.52 3.11 13.93 PMON68537 GM_A36311 70.82 3.97 6.52 3.18 14.29PMON68537 GM_A36311 16.67 9.39 13.65 4.44 54.77 PMON68537 GM_A3631278.32 4.3 6.36 1.79 8.16 PMON68537 GM_A36312 77.55 4.46 6.51 2.13 8.23PMON68537 GM_A36312 77.43 4.17 6.31 1.81 9.24 PMON68537 GM_A36312 76.984.29 6.25 2.27 9.05 PMON68537 GM_A36312 76.43 4.55 6.82 2.16 8.96PMON68537 GM_A36312 76.38 4.5 6.46 2.04 9.54 PMON68537 GM_A36312 75.254.27 6.41 1.97 11.06 PMON68537 GM_A36312 18.24 9.43 13.6 3.07 54.75PMON68537 GM_A36313 80.18 4.07 6.17 2.59 5.85 PMON68537 GM_A36313 79.964.16 6.03 2.59 6.11 PMON68537 GM_A36313 78.88 3.9 5.6 2.8 7.65 PMON68537GM_A36313 78.76 3.92 5.44 2.91 7.82 PMON68537 GM_A36313 77.64 4.22 5.882.9 8.25 PMON68537 GM_A36313 76.15 4.14 6.06 3.13 9.42 PMON68537GM_A36313 19.05 8.87 13.45 3.71 54.03 PMON68537 GM_A36313 18.47 8.4613.13 3.63 55.41 PMON68537 GM_A36314 80.27 3.17 5.77 3.4 6.03 PMON68537GM_A36314 79.66 3.24 5.72 3.19 6.91 PMON68537 GM_A36314 79.5 3.45 5.833.23 6.74 PMON68537 GM_A36314 77.42 3.52 5.76 3.57 8.42 PMON68537GM_A36314 77.33 3.71 6.36 3.34 8.01 PMON68537 GM_A36314 76.83 3.71 6.383.24 8.59 PMON68537 GM_A36314 16.6 9.3 12.63 4.43 55.99 PMON68537GM_A36314 15.26 8.59 13.71 4.54 56.84 PMON68537 GM_A36315 20.21 8.2513.61 3.59 53.37 PMON68537 GM_A36315 17.47 9.22 13.46 3.35 55.57PMON68537 GM_A36315 16.75 9.3 13.61 3.66 55.75 PMON68537 GM_A36315 16.549.18 13.54 3.88 55.9 PMON68537 GM_A36315 16.06 10.07 13.44 4.01 55.42PMON68537 GM_A36315 16.05 9.58 12.82 4.25 56.29 PMON68537 GM_A3631515.95 10.42 13.12 3.63 55.91 PMON68537 GM_A36315 15.5 10.22 13.25 3.7856.3 PMON68537 GM_A36316 79.61 3.56 5.79 2.94 6.87 PMON68537 GM_A3631675.11 4.01 6.45 3.44 9.76 PMON68537 GM_A36316 75.07 4.25 6.74 3.09 9.64PMON68537 GM_A36316 73.92 3.97 6.53 3.56 10.75 PMON68537 GM_A36316 17.269.59 13.1 4.26 54.78 PMON68537 GM_A36316 17.15 9.03 12.81 4.04 55.97PMON68537 GM_A36316 16.62 9.2 13.15 3.99 56.03 PMON68537 GM_A36316 16.69.44 13.19 3.95 55.84 PMON68537 GM_A36317 18.96 7.55 13.2 3.75 55.51PMON68537 GM_A36317 16.19 9.43 13.33 3.96 56.04 PMON68537 GM_A3631716.05 9.1 14.02 3.94 55.91 PMON68537 GM_A36317 15.33 9.4 13.91 4.2256.11 PMON68537 GM_A36317 15.28 9.2 13.87 4.27 56.36 PMON68537 GM_A3631714.58 10.15 13.74 4.38 56.15 PMON68537 GM_A36317 13.95 9.47 13.98 4.7656.79 PMON68537 GM_A36317 13.91 9.88 14.26 4.62 56.25 PMON68537GM_A36318 78.82 3.64 5.7 2.77 7.87 PMON68537 GM_A36318 77.94 3.73 5.92.94 8.29 PMON68537 GM_A36318 75.18 4.11 6.08 3.48 9.95 PMON68537GM_A36318 75.1 3.93 6.02 3.04 10.75 PMON68537 GM_A36318 75.01 4.22 6.573.29 9.72 PMON68537 GM_A36318 74.17 4.2 6.51 3.27 10.68 PMON68537GM_A36318 73.47 4.27 6.7 3.22 11.16 PMON68537 GM_A36318 30.57 10.5414.83 5.55 36.92 PMON68537 GM_A36319 80 3.65 5.83 2.31 7.02 PMON68537GM_A36319 79.89 3.65 5.64 2.35 7.26 PMON68537 GM_A36319 79.4 3.59 5.731.76 8.46 PMON68537 GM_A36319 78 3.87 6.11 2.35 8.5 PMON68537 GM_A3631976.08 4.22 6.5 2.35 9.74 PMON68537 GM_A36319 75.56 3.89 6.41 1.78 11.3PMON68537 GM_A36319 75.26 4.27 6.47 2.37 10.5 PMON68537 GM_A36319 75.164.1 6.48 2.49 10.66 PMON68537 GM_A36320 81.27 3.19 5.84 2.4 6.09PMON68537 GM_A36320 80.21 3.27 5.18 2.44 7.76 PMON68537 GM_A36320 79.643.38 5.5 2.67 7.63 PMON68537 GM_A36320 79.46 3.38 5.82 2.67 7.42PMON68537 GM_A36320 78.5 3.59 6.24 2.49 8 PMON68537 GM_A36320 73.83 3.796.72 2.78 11.74 PMON68537 GM_A36320 73.1 3.95 6.9 2.39 12.48 PMON68537GM_A36320 22.99 8.03 12.19 4.81 50.89 PMON68537 GM_A36324 75.93 3.776.58 2.76 9.76 PMON68537 GM_A36324 75.1 4.05 7.01 2.83 9.8 PMON68537GM_A36324 17.83 8.79 12.78 4.11 55.49 PMON68537 GM_A36324 16.46 8.8812.84 4.48 56.29 PMON68537 GM_A36324 16.35 9.25 13.51 4.17 55.66PMON68537 GM_A36324 15.25 8.99 13.73 4.28 56.69 PMON68537 GM_A3632414.16 10.17 13.95 4.11 56.58 PMON68537 GM_A36324 13.59 9.87 14.61 4.556.33 PMON68537 GM_A36357 80.19 3.03 5.59 3.2 6.62 PMON68537 GM_A3635779.78 3.19 5.51 3.24 6.89 PMON68537 GM_A36357 78.5 3.55 5.75 3.17 7.71PMON68537 GM_A36357 77.48 3.68 5.71 3.55 8.23 PMON68537 GM_A36357 77.283.79 5.66 3.48 8.46 PMON68537 GM_A36357 77.1 3.51 5.43 3.65 8.99PMON68537 GM_A36357 71.9 4.24 6.47 3.67 12.39 PMON68537 GM_A36357 17.669.32 13.26 4.21 54.51 PMON68537 GM_A36359 77.91 3.35 5.67 3.24 8.53PMON68537 GM_A36359 77.85 3.29 5.42 3.29 8.87 PMON68537 GM_A36359 76.713.65 6.07 3.35 8.95 PMON68537 GM_A36359 71.73 4.01 6.79 3.49 12.68PMON68537 GM_A36359 69.32 4.51 6.99 3.66 14.13 PMON68537 GM_A36359 68.634.44 6.91 3.76 14.89 PMON68537 GM_A36359 18.87 8.03 13.38 3.86 54.81PMON68537 GM_A36359 16.81 9.83 13.08 4.68 54.55 PMON68537 GM_A3636079.34 3.29 5.99 3.15 6.88 PMON68537 GM_A36360 75.42 3.47 6.47 3.08 10.26PMON68537 GM_A36360 75.3 3.86 6.69 3.2 9.64 PMON68537 GM_A36360 74.513.8 6.39 3.32 10.67 PMON68537 GM_A36360 21.49 6.95 13.07 3.92 53.46PMON68537 GM_A36360 20.05 7.4 13.09 3.83 54.57 PMON68537 GM_A36360 16.089.14 13.02 4.64 56.03 PMON68537 GM_A36360 15.86 9.07 13.44 4.49 56.04PMON68537 GM_A36361 82.13 2.83 5.67 3.13 4.81 PMON68537 GM_A36361 80.993.2 5.79 3.01 5.64 PMON68537 GM_A36361 74.39 3.85 6.33 3.5 10.59PMON68537 GM_A36361 18.01 8.46 13.18 3.92 55.41 PMON68537 GM_A3636117.99 8.11 13.05 4.09 55.7 PMON68537 GM_A36361 17.35 8.31 13.4 4 55.88PMON68537 GM_A36361 16.81 10.2 12.9 4.32 54.87 PMON68537 GM_A36361 16.558.5 13.21 4.22 56.45 PMON68537 GM_A36362 78.05 3.89 6.29 2.81 7.76PMON68537 GM_A36362 76.89 3.69 6.32 3.12 8.76 PMON68537 GM_A36362 76.1 46.57 3.02 9.24 PMON68537 GM_A36362 76.01 4.08 6.24 3.03 9.48 PMON68537GM_A36362 75.86 3.76 5.68 3.56 9.95 PMON68537 GM_A36362 75.79 4.07 6.433.15 9.34 PMON68537 GM_A36362 74.89 4.14 6.63 3.11 10.07 PMON68537GM_A36362 17.22 8.8 13.75 3.77 55.54 PMON68537 GM_A36363 79.15 3.57 6.23.03 6.84 PMON68537 GM_A36363 75.69 3.83 7.07 2.73 9.53 PMON68537GM_A36363 73.97 4.22 6.82 3.39 10.33 PMON68537 GM_A36363 72.53 4.31 6.643.7 11.59 PMON68537 GM_A36363 68.42 4.5 7.05 3.95 14.79 PMON68537GM_A36363 18.39 8.7 13.61 4.1 54.28 PMON68537 GM_A36363 17.54 8.87 14.084.07 54.56 PMON68537 GM_A36363 15.87 9.66 14.56 4.2 54.69 PMON68537GM_A36365 78.79 3.11 5.87 1.27 9.9 PMON68537 GM_A36365 76.76 3.86 5.791.66 10.91 PMON68537 GM_A36365 75.41 3.49 6.06 1.83 12.15 PMON68537GM_A36365 73.57 3.65 6.11 1.5 14.19 PMON68537 GM_A36365 71.55 3.56 6.621.24 16.08 PMON68537 GM_A36365 70.41 4 6.07 2.15 16.33 PMON68537GM_A36365 66.66 3.9 6.84 1.5 20.21 PMON68537 GM_A36365 63.96 4.22 7.082.27 21.52 PMON68537 GM_A36366 75.44 4.33 6.49 3.21 9.32 PMON68537GM_A36366 74.75 4.21 6.87 2.71 10.33 PMON68537 GM_A36366 74.69 4.65 6.913.06 9.65 PMON68537 GM_A36366 73.23 4.89 7.23 2.99 10.52 PMON68537GM_A36366 72.53 4.76 7.42 3.26 10.85 PMON68537 GM_A36366 67.15 5.05 7.473.33 15.87 PMON68537 GM_A36366 65.81 5.6 7.9 3.37 16.09 PMON68537GM_A36366 62.31 6.19 8.71 3.22 18.55 PMON68537 GM_A36367 80.56 3.3 6.072.58 6.34 PMON68537 GM_A36367 77.78 3.58 6.47 2.66 8.45 PMON68537GM_A36367 77.78 3.46 6.25 2.84 8.51 PMON68537 GM_A36367 77.39 3.81 6.712.86 8.11 PMON68537 GM_A36367 77.32 3.74 6.17 3.12 8.47 PMON68537GM_A36367 75.93 3.97 6.23 3.43 9.29 PMON68537 GM_A36367 72.82 4.09 6.853.25 11.88 PMON68537 GM_A36367 19.31 7.58 13.7 3.59 55 PMON68537GM_A36410 21.67 7.62 13.38 3.43 53.1 PMON68537 GM_A36410 20.9 8.33 12.933.64 53.33 PMON68537 GM_A36410 20.21 8.04 13.28 3.86 53.66 PMON68537GM_A36410 20.02 8.71 12.79 3.71 53.87 PMON68537 GM_A36410 18.96 8.9513.3 3.77 54.15 PMON68537 GM_A36410 18.18 8.98 13.56 3.74 54.66PMON68537 GM_A36410 17.61 9.29 12.93 4.12 55.13 PMON68537 GM_A3641016.78 9.8 13.78 3.92 54.83 PMON68537 GM_A36411 75.06 4.33 6.49 2.9310.08 PMON68537 GM_A36411 74.32 4.46 6.76 2.96 10.38 PMON68537 GM_A3641173.41 4.76 6.91 3.11 10.78 PMON68537 GM_A36411 73.24 4.87 7.28 2.8910.67 PMON68537 GM_A36411 22.38 8.17 13.47 3.6 51.51 PMON68537 GM_A3641118.26 9.07 14.14 3.81 54.02 PMON68537 GM_A36411 17.52 10.1 13.1 4.0354.36 PMON68537 GM_A36411 17.02 9.71 13.45 4.02 54.89 A3244 A3244 18.297.79 13.69 4.15 55.08 A3244 A3244 17.54 8.19 13.32 4.32 55.57 A3244A3244 17.13 8.13 13.21 4.46 56.04 A3244 A3244 15.47 9.56 13.04 4.4356.46 A3244 A3244 15.17 8.95 13.79 4.3 56.78 A3244 A3244 15.05 9.0314.16 4.01 56.8 A3244 A3244 13.51 10.07 12.95 5.07 57.3 A3244 A324413.49 9.91 13.31 4.56 57.67

Example 6 FAD2-1/FATB dsRNAi Construct in Transgenic Soybean

Construct pMON95829 as described in Example 3D is used to introduce aFAD2-1 intron, FATB, double-stranded RNA-forming construct into soybeanfor suppressing the FAD2 gene and FATB genes. The vector is stablyintroduced into soybean (Asgrow variety A4922) via Agrobacteriumtumefaciens strain ABI (Martinell, U.S. Pat. No. 6,384,301). The CP4selectable marker allows transformed soybean plants to be identified byselection on media containing glyphosate herbicide. Subsequently, thegenomes of transformed plants are screened for concurrent tandeminsertion of the first T-DNA and the second T-DNA, i.e. in the “rightborder to right border” assembly. Screening is done with Southernhybridization mapping methods. Transformed soybean plants containing thepreferred configuration in their genome are transferred to a green housefor seed production.

For example, leaf tissue was taken from the R0 plants transformed withconstruct pMON95829 and Southern analysis is performed. Probes andrestriction enzyme digests are chosen in order to identify eventscontaining a right-border-right-border (“RB-RB”) assembly of bothT-DNAs. Typically, approximately 25% of all transformants have properlyassembled RB-RB T-DNAs.

Fatty acid compositions are analyzed from seed of soybean linestransformed with a pMON95829 construct using gas chromatography asdescribed in Example 4 to identify methyl esters of fatty acid compoundsextracted from seeds. First, six R1 seeds taken from soybean plantstransformed with construct pMON95829 are harvested, and the fatty acidcomposition of each single seed is determined. Since R1 plants of eachevent are segregating for the transgenes and, therefore, yield seedswith conventional soybean composition, as well as modified versions. Thepositive seeds are pooled and averaged for each event. The pooledpositive averages demonstrate that the mono- and polyunsaturated fattyacid compositions are altered in the oil of seeds from transgenicsoybean lines as compared to that of the seed from non-transformedsoybean (See Table 4). For example, FAD2 suppression provides plantswith increased amount of oleic acid ester compounds and FATB suppressionprovides plants with reduced saturated fatty ester compounds, e.g.palmitates and stearates.

TABLE 4 Fatty acid composition of R1 single seeds from pMON95829 events.Construct Event # 16:0 18:0 18:1 18:2 18:3 PMON95829 GM_A94247 2.1 2.883.0 6.0 5.5 PMON95829 GM_A94296 2.6 2.9 80.6 7.1 5.8 PMON95829GM_A93590 2.5 2.8 80.4 7.4 5.8 PMON95829 GM_A93437 2.6 2.8 79.8 7.9 6.0PMON95829 GM_A93517 2.9 2.8 79.5 7.7 6.0 PMON95829 GM_A93647 2.3 3.078.6 9.0 6.5 PMON95829 GM_A93670 3.1 2.9 77.3 10.1 6.2 PMON95829GM_A92396 2.9 2.6 76.0 11.1 7.0 PMON95829 GM_A92455 3.6 3.1 74.9 12.05.5 PMON95829 GM_A93678 2.8 3.4 74.0 11.9 7.4 PMON95829 GM_A93640 2.52.7 71.6 14.6 7.6 PMON95829 GM_A94937 4.5 3.3 67.2 17.7 7.1 PMON95829GM_A92481 4.9 2.8 58.1 25.3 8.1 PMON95829 GM_A94306 3.1 3.2 55.9 29.07.9 PMON95829 GM_A94211 3.0 2.7 47.0 38.3 8.7

Example 7

pMON93505 is a construct used for in vivo assembly and has two T-DNAsegments, each flanked by Agrobacterium T-DNA border elements, i.e.right border DNA (RB) and left border DNA (LB). The first T-DNA segmentcontains a soybean 7Sα′ promoter operably linking to a soybean FAD2-1Aintron 1 (SEQ ID NO: 1), which is reduced by 100 contiguous nucleotidesfrom the 3′ end and ligated to the FATB-1a3′ UTR followed by a FATB-1a5′UTR, a C. pulcherrima KAS IV gene (SEQ ID NO: 39) operably linking to aBrassica napin promoter and a Brassica napin 3′ termination sequence, aRicinus communis delta 9 desaturase gene (U.S. Patent ApplicationPublication No. 2003/00229918 A1, now U.S. Pat. No. 7,078,588) operablylinking to a soybean 7Sα′ promoter and a nos 3′ termination sequence,and a CP4 EPSPS gene operably linking to an eFMV promoter and a peaRubisco E9 3′ termination sequence all flanked by Agrobacterium T-DNAborder elements, i.e. right border DNA (RB) and left border DNA (LB). Onthe same construct, in the second T-DNA segment, flanked by another RBand LB, is a H6 3′ termination sequence operably linking to a soybeanFAD2-1A intron 1 (SEQ ID NO: 1), which is reduced by 100 contiguousnucleotides from the 3′ end and ligated to the FATB-1A 3′ UTR followedby a FATB-1a 5′ UTR.

Construct pMON93505 is stably introduced into soybean (Asgrow varietyA4922) via Agrobacterium tumefaciens strain ABI (Martinell, U.S. Pat.No. 6,384,301). The CP4 selectable marker allows transformed soybeanplants to be identified by selection on media containing glyphosateherbicide. Subsequently, the genomes of transformed plants are screenedfor concurrent tandem insertion of the first T-DNA and the second T-DNA,i.e. in the “right border to right border” assembly. Screening is donewith Southern hybridization mapping methods. Transformed soybean plantscontaining the preferred configuration in their genome are transferredto a green house for seed production.

For example, leaf tissue was taken from the R0 plants transformed withconstruct pMON93505 and Southern analysis is performed. Probes andrestriction enzyme digests are chosen in order to identify eventscontaining a right-border-right-border (“RB-RB”) assembly of bothT-DNAs. Typically, approximately 25% of all transformants have properlyassembled RB-RB T-DNAs.

Fatty acid compositions are analyzed from seed of soybean linestransformed with a pMON93505 construct using gas chromatography asdescribed in Example 4 to identify methyl esters of fatty acid compoundsextracted from seeds. First, six R1 seeds taken from soybean plantstransformed with construct pMON93505 are harvested, and the fatty acidcomposition of each single seed is determined. Since R1 plants of eachevent are segregating for the transgenes and, therefore, yield seedswith conventional soybean composition, as well as modified versions. Thepositive seeds are pooled and averaged for each event. The pooledpositive averages demonstrate that the mono- and polyunsaturated fattyacid compositions are altered in the oil of seeds from transgenicsoybean lines as compared to that of the seed from non-transformedsoybean (See Table 5). For example, FAD2 suppression provides plantswith increased amount of oleic acid ester compounds. For instance, FAD2suppression provides plants with increased amount of oleic acid estercompounds, FAD3 suppression provides plants with decreased linolenicacid ester compounds, and FATB suppression provides plants with reducedsaturated fatty ester compounds, e.g. palmitates and stearates.

TABLE 5 Fatty acid composition of R1 single seeds from pMON93505 eventsConstruct Event # 16:0 18:0 18:1 18:2 18:3 PMON93505 GM_A87814 1.3 1.084.9 5.5 6.3 PMON93505 GM_A86449 1.5 0.9 84.9 4.9 6.8 PMON93505GM_A86032 1.5 1.1 83.5 6.3 7.0 PMON93505 GM_A86159 1.5 0.9 82.8 6.7 7.5PMON93505 GM_A86178 1.7 1.0 82.5 6.7 7.3 PMON93505 GM_A86075 1.4 0.981.4 6.6 8.5 PMON93505 GM_A86303 1.0 0.6 81.4 7.4 8.8 PMON93505GM_A86454 1.4 0.9 79.9 7.4 8.8 PMON93505 GM_A86799 1.4 1.1 79.4 9.6 7.7PMON93505 GM_A85997 2.2 2.5 79.3 7.7 7.4 PMON93505 GM_A86058 1.8 1.076.8 11.3 8.3 PMON93505 GM_A86274 1.2 0.7 74.6 10.2 11.9 PMON93505GM_A86325 1.1 0.7 72.8 15.4 9.2 PMON93505 GM_A85969 2.0 0.7 70.7 13.612.1 PMON93505 GM_A86033 1.7 0.9 69.1 18.2 9.5 PMON93505 GM_A86372 1.71.0 65.7 12.6 17.6 PMON93505 GM_A86403 1.5 0.9 64.6 16.8 15.4 PMON93505GM_A87803 1.1 0.6 57.7 26.0 13.8 PMON93505 GM_A86036 3.1 1.5 54.8 30.49.7 PMON93505 GM_A86269 4.9 1.8 51.4 31.9 9.5

Example 8 Transgenic Soybeans with Altered Fatty Acid Compositions

pMON97563 contains a soybean 7Sα′ promoter operably linked to a soybeanFAD2-1A intron 1 (SEQ ID NO: 1), which is reduced by 100 contiguousnucleotides from the 3′ end and linked to a FAD3-1A 5′UTR, followed by aFAD3-1A 3′UTR, linked to a FAD3-1B 5′UTR, followed by a FAD3-1B 3′UTR,linked to a FAD3-1C5′UTR, followed by a FAD3-1C3′UTR, followed by aFATB-1a CTP coding region, followed by a FATB-2a CTP coding regionoperably linking to 70 nucleotides from FAD3-1A intron 4, operablylinking to a FATB-2a CTP coding region in the anti-sense orientationfollowed by a FATB-1a CTP coding region in the antisense orientation,linked to a FAD3-1C 3′UTR in antisense, followed by a FAD3-1C5′UTR inantisense, linked to a FAD3-1B 3′UTR in antisense, followed by a FAD3-1B5′UTR in antisense, linked to a FAD3-1A 3′UTR in antisense, followed bya FAD3-1A 5′UTR in antisense, followed by a soybean FAD2-1A intron 1(SEQ ID NO: 1), which is reduced by 400 contiguous nucleotides from the3′ end and in the anti-sense orientation, operably linked to a H6 3′polyadenylation segment with a CP4 EPSPS gene operably linking to aneFMV promoter and a pea Rubisco E9 3′ termination sequence all flankedby RB and LB on the same DNA molecule. The resulting gene expressionconstruct is used for plant transformation using methods as describedherein. Fatty acid compositions are determined from seed of soybeanlines transformed with this construct using gas chromatography asdescribed in Example 4. Table 6 gives the compositions of representativeseeds. The level of 18:3 is reduced to approximately 1%.

TABLE 6 Fatty acid composition of R1 single seeds from pMON97563 eventsConstruct Event 16:0 18:0 18:1 18:2 18:3 PMON97563 GM_A109156 2.21 2.7885.05 8.48 0.69 PMON97563 GM_A109196 2.07 2.31 84.4 9.42 0.97 PMON97563GM_A109207 2.24 2.78 83.98 9.36 0.82 PMON97563 GM_A103543 2.21 2.6383.94 10.28 0.95 PMON97563 GM_A103547 2.06 2.47 83.67 10.47 0.89PMON97563 GM_A109146 1.71 2.34 81.14 13.71 0.91 PMON97563 GM_A1091552.33 2.7 80.76 12.28 1.11 PMON97563 GM_A109164 2.07 2.61 78.8 14.6 1PMON97563 GM_A109170 2.68 1.95 78.78 14.14 1.55 PMON97563 GM_A1092772.49 3.19 78.19 14.51 0.93 PMON97563 GM_A109194 2.46 2.81 76.62 16.260.92 PMON97563 GM_A109177 2.56 2.49 72.64 20.14 1.44 PMON97563GM_A109201 2.46 2.9 72.21 20.13 1.11 PMON97563 GM_A103550 2.18 2.6770.84 22.25 1.17 PMON97563 GM_A109203 2.18 2.81 69.93 22.91 0.98

Example 9 Crosses of Mid-Oleic Transgenic Soybean with Low LinolenicSoybean

A soybean plant of a line with seeds having mid-oleic acid levels in itsoil is crossed with a plant from a line with normal oleic acid levelsbut about 2.8% linolenic acid (18:3). This cross results in a soybeanline producing oil with the combined properties, mid-oleic acid and lowlinolenic acid.

Briefly, plant breeding was performed as described below. One parentline, a transgenic soybean line, labeled event GM_A22234, contains theplasmid pMON68504 in a chromosome. pMON68504 is a 2T-DNA constructhaving a 7S promoter operably linked to a FAD2-1A intron #1 (SEQ ID NO:2; PCT Publication WO 2001014538) in sense orientation in order topartially suppress the endogenous FAD2 gene and a CP4 selectable markergene. The oil extracted from the seeds of this line containsapproximately 65% oleic acid, up from the 20% of conventional soybeanoil (see Table 7). Another parent line is a non-transgenic variety6p248-5 (C1640 line) which has a linolenic acid content of about 3% byweight of total fatty acids in its seeds, as compared to theconventional 8-9% linolenic acid found in normal soybean oil (see Table7). The reduction in linolenic acid is caused by afad3-1b-/fad3-1c-double mutant. (See Wilcox, J. R. and J. F. Cavins,Inheritance of low linolenic acid content of the seed of a mutant ofGlycine max., Theoretical and Applied Genetics 71: 74-78, 1985).

Plants of the transgenic line GM_A22234 (used as female) and the mutantline 6p248-5 (used as the male) were crossed. Thirty F1 seeds wereproduced and planted to produce 2.3 lbs of selfed F2 seeds. Putativetriple homozygous seeds were identified from 200 F2 seeds through singleseed fatty acid methyl-ester (FAME) analysis of seed chips. Twenty-sevenseeds with about 60% 18:1, about 20% 18:2, and about 2-3% 18:3 wereidentified and planted to produce selfed F3 seeds.

For marker analysis, F2 leaf tissue samples were collected andestablished molecular markers for the FAD3 mutant alleles were used toidentify double positive plants (plants having both FAD3-1B and FAD3-1Cmutations). Three genotypes were targeted for recovery from thisexperiment: 1) fad3-1b-/fad3-1c-double homozygous mutants; 2) singlehomozygous plants for the fad3-1c-allele alone; and 3) single homozygousfor fad3-1b-allele alone.

The F2 plants were single plant harvested, and 10 F3 seed sub-sampleswere analyzed. From 27 seeds with about 60% 18:1 (oleic acid), about 20%18:2 (linoleic acid), and about 2-3% 18:3 (linolenic acid), 5 plantswere identified as putative double-FAD3 mutant and were bulked togetherfor further growth. Table 7 summarizes the F3 seed composition data from120 F2 plants.

TABLE 7 Mid-oleic acid phenotype/fad3 mutant stack- F3 seed fatty acidcomposition Fatty acid, Relative mole % GOI 18:1 16:0 18:0 18:2 18:3mid-oleic(GM_A22234), fad3-1b-, 74.3 9.08 3.65 7.89 1.91 Fad3-1c-(6p248-5) fad3-1b-, Fad3-1c- Mutant Parent 30.5 12.3 3.61 50.90 2.3(6p248-5) ~65% mid-oleic Parent 64.6 9.4 3.61 14.53 7.27 (GM_A22234)

The triple fad3-1b-, fad3-1c-, mid-oleic acid line (GM_AA22234) has 1.9%18:3 linolenic and 74.3% of oleic acid. The combination of fad3-1b- andfad3-1c-mutants with the transgenic mid-oleic (GM_AA22234) locus leadsto further reduction of linolenic and increase of oleic relative to therespective parent lines.

To evaluate the field efficacy of the triple fad3-1b-, fad3-1c-,mid-oleic (GM_AA22234) line, the breeding stack entries were planted ina group block design with the stacks and parental controls grouped andrandomized within the testblock, and seed samples were analyzed. A fattyacid profile for the triple fad3-1b-, fad3-1c-, mid-oleic (GM_AA22234)stack was generated with F4 field grown seed using single seed FAME. F4fatty acid profile demonstrated approximately 68% 18:1, 13% totalsaturates, 16% 18:2 and 2.3% 18:3. Oil and protein levels were similarto the parental lines.

Example 10 Crosses of Mid-Oleic, Low Saturate Transgenic Soybean withLow Linolenic Soybean

A soybean plant of a line with seeds having mid-oleic acid and lowsaturates level in its oil is crossed with a plant from a line withnormal oleic and saturate levels but about 2.8% linolenic acid (18:3).This cross results in a soybean line producing oil with the combinedproperties, mid-oleic, low saturated and low linolenic fatty acidlevels.

Briefly, plant breeding was performed as described below. One parentline is a transgenic soybean line harboring recombinant DNA for partialsuppression of the endogenous genes FAD2-1 and FATB as well as a CP4selectable marker gene which renders the plant tolerant to glyphosate.The oil extracted from the seeds of this line contains approximately55-85% oleic acid, up from the 20% of conventional soybean oil. It alsocontains less than 8% saturated fatty acids (16:0 plus 18:0), reducedfrom the conventional 14-16% of normal soybean oil. Another parent lineis a non-transgenic variety 6p248-5 (C1640 line) which has about 3%linolenic acid levels in its seeds, as compared to the conventional 8-9%linolenic acid found in normal soybean oil. The reduction in linolenicacid is caused by a fad3-1b-/fad3-1c-double mutant. (See Wilcox, J. R.and J. F. Cavins, Inheritance of low linolenic acid content of the seedof a mutant of Glycine max., Theoretical and Applied Genetics 71: 74-78,1985.)

Plants of the transgenic mid-high oleic/low saturate line are crossedwith plants from the mutant line 6p248-5. F1 seeds are produced andplanted to produce selfed F2 seed. Putative triple homozygous seeds areidentified from F2 seeds through single seed fatty acid methyl-ester(FAME) analysis of seed chips. Seeds with combined oil traits areidentified and planted to produce selfed F3 seeds. For marker analysis,F2 leaf tissue samples are collected and established molecular markersfor the FAD3 mutant alleles are used to identify double positive plants(plants having both FAD3 deletions). F3 seed lots which indicatehomozygosity for the transgene locus as well as the two FAD3 mutationsare selected and used for line establishment.

To evaluate the field efficacy of the fad3-1b-, fad3-1c-, mid-oleic/lowsat lines, the breeding stack entries are planted in a group blockdesign with the stacks and parental controls grouped and randomizedwithin the test block, and seed samples are analyzed. A fatty acidprofile for the triple fad3-1b-, fad3-1c-, mid-oleic/low sat stack isdetermined with F4 field grown seed using single seed FAME. F4 fattyacid profile shows 55-85% 18:1, less than 8% saturates, and 2-3% 18:3.Oil and protein levels are similar to the parental lines.

Example 11 Use of Polymorphisms at FAD3-1b

To practice the methods of the invention, polymorphisms associated withthe soybean FAD3-1B gene can be used to identify the presence of soybeangenomic regions associated with certain low linolenic acid phenotypes. Asingle nucleotide polymorphism at a position corresponding to position2021 of SEQ ID NO:61 is detected among all the lines in an entiresequence length of 2683 bp (Table 8) and is associated with alow-linolenic acid phenotype. Low-linolenic lines 6P248, T27111, T27190,T26767 and T26830 carry a “T” allele at this position while all otherlines carry a “C”. Consequently, the presence of a “T” allele can beused to identify the presence of the low linolenic soybean genomicregions in crosses where low linolenic germplasm derived from 6P248,T27111, T27190, T26767 and T26830 are used. Other low-linolenic linessuch as A5, Soyola, and N98-4445 carry a wild type allele at this locus,indicating that one or more other loci contribute to the low-linolenicphenotype in the A5, Soyola, and N98-4445 lines.

TABLE 8 Polymorphisms at the FAD3-1B locus LinesPosition 2021 of SEQ ID NO: 61 Orig seq C 6P248 T T27111 T T27190 TT26767 T T26830 T A5 C C1640 C Soyola C N98-4445 C A2247 C AG1701 CAG1902 C AG2402 C AG2703 C AG3201 C AG3302 C AG3702 C AJB2102J0C CAJB2302K0C C CSR2533 C CSR2622N C CSR3922N C DKB19-51 C DKB23-95 CWP25920 C

Example 12 Identification of Polymorphisms in the FAD3-1C Gene

To practice the methods of the invention, polymorphisms associated withthe soybean FAD3-1C gene are used to identify the presence of soybeangenomic regions associated with certain low linolenic acid phenotypes.Four SNPs and one indel (insertion/deletion) are identified at FAD3-1Cthat are associated with certain low linolenic acid phenotypes (Table9). The SNPs corresponding to positions 687, 2316, 3743, as well as theindel at 1129 of SEQ ID NO:62 are associated with the low-linolenicphenotype. Low-linolenic lines, Soyola and N98-4445 carry a differentallele at positions 687 and 1129 from all the other lines.

Mutant lines 6P248, T27111, T27190, T26767, T26830 and A5 will fail toamplify with certain FAD3-1C locus-specific primers as there is a largedeletion at the FAD3-1C locus in these lines. The failure of theseregions to be amplified, coupled with appropriate positive controlreactions (i.e. using soybean genomic DNA that contains an intactFAD3-1C gene with FAD3-1C primers from the deleted region as well as useof primers to other non-FAD3-1C genes with the soybean genomic DNA fromthe FAD3-1C deletion), is diagnostic for FAD3-1C deletions.

TABLE 9 Polymorphisms at the FAD3-1C locus Sequence position Lines 6871129 1203 2316 3292 3360 3743 6P248 NA NA NA N/A T27111 NA NA NA N/AT27190 NA NA NA N/A T26767 NA NA NA N/A T26830 NA NA NA N/A A5 NA NA NAT C1640 T * A Soyola C T A T C A A N98-4445 C T A A2247 T * A G T * *AG1701 T * A G T * * AG1902 T * A T * * AG2402 T * A G T * * AG2703 T *A AG3201 T * G AG3302 T * A AG3702 T * A AJB2102J0C T * A AJB2302K0C T *A CSR2533 T * A CSR2622N T * G CSR3922N T * A DKB19-51 T * A DKB23-95T * A WP25920 T * A Note: 1. NA means no amplification

Example 13 Identification of Soybean FAD3-1C Promoter Polymorphisms

To practice the methods of the invention, polymorphisms associated withthe soybean FAD3-1C promoter are used to identify the presence ofsoybean genomic regions associated with certain low linolenic acidphenotypes. As noted in Table 10, low linolenic lines Soyola andN98-4445 carried a different allele at all seven positions from theother wild-type lines. The presence of these polymorphisms could be usedto identify the presence of Soyola or N98-4445 germplasm in crosses towild type germplasm.

TABLE 10 Polymorphisms at FAD3-1C Promoter Region Position 334 364 385387 393 729 747 Soyola G C T A C G C N98-4445 G C T A C G C Wildtypes AG G T T T T (16 lines)

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. Soybean seed, said seed having a fatty acidcomposition comprising a linolenic acid content of 0.5% to 3% of totalseed fatty acids by weight, a saturated fatty acid content of 10% to 15%of total seed fatty acids by weight, and an oleic acid content of 70% to79% of total seed fatty acids by weight, a transgene that decreases theexpression of an endogenous soybean FAD2-1 gene, and at least oneloss-of-function mutation in an endogenous soybean FAD3 gene.
 2. Thesoybean seed of claim 1, said seed having a fatty acid compositioncomprising a linolenic acid content of 1% to 3% of total seed fattyacids by weight and an oleic acid content of 70% to 75% of total seedfatty acids by weight.
 3. The seed of claim 2, wherein the linolenicacid content is 1% to 2%.
 4. The soybean seed of claim 1, wherein saidseed comprises at least two loss-of-function mutations in at least twoendogenous soybean FAD3 genes.
 5. The soybean seed of claim 4, whereinsaid endogenous soybean FAD3 genes are FAD3-1B and FAD3-1C.
 6. Thesoybean seed of claim 4, wherein at least one of said loss-of-functionmutations comprises a deletion in the FAD3-1C gene of SEQ ID NO:62. 7.The soybean seed of claim 1, wherein said transgene that decreases theexpression of an endogenous soybean FAD2-1 gene comprises a FAD2 geneelement of about 50, 75, 100, 150, 175, 200, 220, 250, 300, 320, 350,400, 420, 450, 500, 550, 600, 800, or 1000 nucleotides in length.
 8. Theseed of claim 1, wherein the linolenic acid content is 0.5% to 2%. 9.The seed of claim 1, wherein the linolenic acid content is 1% to 2%. 10.The seed of claim 1, wherein said seed are in a container containinggreater than 500 of said seed.
 11. The seed of claim 10, wherein saidseed are homozygous for the transgene and homozygous for at least oneloss of function mutation in the endogenous FAD3 gene.
 12. Soybean seed,said seed having a fatty acid composition comprising a linolenic acidcontent of about 0.5% to 3% of total seed fatty acids by weight, asaturated fatty acid content of 2% to 8% of total seed fatty acids byweight, and an oleic acid content of 70% to 79% of total seed fattyacids by weight, at least one transgene that decreases the expression ofboth an endogenous soybean FAD2-1 and an endogenous FATB gene, and atleast one loss-of-function mutation in an endogenous soybean FAD3 gene.13. The seed of claim 12, said seed having a fatty acid compositioncomprising a linolenic acid content of about 1% to 3% of total seedfatty acids by weight, a saturated fatty acid content of about 2% to 8%of total seed fatty acids by weight, and an oleic acid content of 70% to75% of total seed fatty acids by weight.
 14. The seed of claim 13, thelinolenic acid content is 1% to 2%.
 15. The seed of claim 12, whereinsaid seed comprises at least two loss-of-function mutations in at leasttwo endogenous soybean FAD3 genes.
 16. The seed of claim 15, whereinsaid endogenous soybean FAD3 genes are FAD3-1B and FAD3-1C.
 17. The seedof claim 12, wherein said loss-of-function mutation comprises a deletionin the FAD3-1C gene of SEQ ID NO:62.
 18. The seed of claim 12, whereinsaid transgene that decreases the expression of an endogenous soybeanFAD2-1 gene comprises a FAD2 gene element of about 50, 75, 100, 150,175, 200, 220, 250, 300, 320, 350, 400, 420, 450, 500, 550, 600, 800, or1000 nucleotides in length.
 19. The seed of claim 12, wherein saidtransgene that decreases the expression of an endogenous soybean FATBgene comprises a FATB gene element of about 50, 75, 100, 150, 175, 200,220, 250, 300, 320, 350, 400, 420, 450, 500, 550, 600, 800, or 1000nucleotides in length.
 20. The seed of claim 12, the linolenic acidcontent is 0.5% to 2%.
 21. The seed of claim 12, wherein the linolenicacid content is 1% to 2%.
 22. The seed of claim 12, wherein said seedare in a container containing greater than 500 of said seed.
 23. Theseed of claim 22, wherein said seed are homozygous for the transgene andhomozygous for at least one loss of function mutation in the endogenousFAD3 gene.